DOI: 10.5061/dryad.msbcc2ftc
title: A practical approach to functional optical coherence tomography shows abnormal retinal responses in Alzheimer’s disease

description: Spectral-domain optical coherence tomography (SD-OCT) is an accessible
 clinical tool for measuring structural changes to the retina, and
 increasingly as a biomarker for brain-predominant neurodegenerative
 diseases like Alzheimer’s. Information about retinal function can also be
 extracted from OCT images, but is under-studied, with literature examples
 often employing challenging protocols or requiring specialized hardware.
 The first goal of this study was to verify that functional retinal imaging
 was feasible with a commercially-available SD-OCT device and a clinically
 practical protocol. Inspired by methods from other functional imaging
 modalities, we acquired images while repeatedly cycling lights on and off,
 and spatially normalized retinas to facilitate intra- and inter-individual
 analyses. In eight healthy young adults, light-dependent increases in
 reflectivity were easily demonstrated at photoreceptor inner and outer
 segments, changing by ~7% in bright light and ~3% in dim light. Bright
 light elicited a subtle (~2%) but consistent light-dependent decrease in
 reflectivity through much of the rest of the retina, including the
 avascular outer nuclear layer (ONL). We speculated that some of these
 changes are influenced by glial function – as through water management – a
 topic of high interest in neurodegenerative diseases that may involve the
 glymphatic system. Functional abnormalities in patients with antibodies
 against aquaporin-4 (n=3) supported this interpretation. We next compared
 patients with early-onset Alzheimer’s disease (n=14) to age-matched
 controls (n=14), revealing that patients had a relatively exaggerated
 light-induced change in ONL reflectivity (p&lt;0.05). Because these
 measurements can be obtained within thirty minutes, regular use in
 research and limited clinical settings is feasible.
description: COLLECTION:   Images were collected on a
 Heidelberg Engineering SPECTRALIS OCT. In Experiments 1, 3, and 4, The
 SPECTRALIS Slice Planning Tool was used to select a single slice of the
 retina per subject, which was automatically re-selected for each
 acquisition. That single slice spanned 30° eccentricity, including and
 angled through the (para-)fovea and the center of the optic disc,
 generating the cross-sectional view seen in Figure 1 of the manuscript.
 Automatic Real Time (ART) averaging was set to the maximum (100). Images
 were collected after &gt; 20 seconds of exposure to light
 ("_LIGHT"), or after 2 minutes of darkness ("_DARK").
 We cycled through light and dark repeatedly, collecting up to fifteen
 images per participant per condition. In Experiment 2, two perpendicular
 slices were collected. Both spanned 30° eccentricity. Due to software
 limitations, neither the horizontally-oriented nor vertically-oriented
 slice could be tilted to cross the fovea and center of the optic disc.
 However, the horizontally-oriented slice approximates the slice position
 used in other experiments.   PROCESSING:
   Raw data were extracted from .sdb files using ImageJ
 (v.1.47)(Import&gt;&gt;RAW&gt;&gt;16-bit unsigned, with
 little-endian byte order). Retinas were cropped from raw reflectivity
 maps, resampled from the native image resolution to isometric pixels (3.89
 μm × 3.89 μm). In experiments using a bright stimulation light, these
 images were saved in the ANALYZE format, named according to the convention
 "[SubjectID]_DARK" or  "[SubjectID]_LIGHT" and are
 readily viewed by dragging and dropping into ImageJ. For young adults
 (SubjectID starts with "YA"), additional images were collected
 under a dim stimulation light, denoted by the suffix "-dim".
 Pixel values in these images are directly proportional to
 reflectivity.   Next, the approximate position of the
 following structures was manually marked: the border between the vitreous
 and the retina, the border between the retinal nerve fiber layer (RNFL)
 and the ganglion cell layer (GCL), the border between the inner plexiform
 layer (IPL) and the inner nuclear layer (INL), the border between the
 outer plexiform layer (OPL) and the typically hyporeflective band
 containing both the Henle fiber layer (HFL) and the outer nuclear layer
 (ONL), the outer limiting membrane (OLM; also called the external limiting
 membrane), and the RPE. Raw and manually-marked data were saved in the
 ANALYZE format for later processing using a custom R (v.3.3.3) script. In
 each subject's folder, ANALYZE images with the "_MARKED"
 suffix denote those with manually-labeled layers. The R script and an
 analysis example are provided in the folder
 "_PROCESSING_EXAMPLE01".   The following
 steps are performed by the custom R script: First, the apparent angle of
 the retina was measured for later use. This value reports on entrance
 position of the OCT machine’s infrared beam, which impacts the angle at
 which it encounters the retina. For the Henle fiber layer in particular,
 even small changes in retinal angle (in the “bright” Experiment 1 data for
 these young adults, 25th percentile, median, and 75th percentile were
 respectively -0.83°, 0.90° and 2.72°, where positive values reflect a
 counter-clockwise rotation from horizontal), can significantly impact
 reflectivity. Next, retinas were linearized based on the contours of the
 RPE. These ANALYZE images are stored with the prefix "_flat_".
 In these flattened retinas, the position of aforementioned
 layers/structures was automatically refined based on local signal
 features. Subsequent steps were applied to a large span of the retina that
 was maximally illuminated when the stimulus light was “on”: Measuring
 along the RPE, this spanned 500 μm to 2750 μm away from the center of the
 fovea, towards the optic nerve head. Since layer thickness varies between
 subjects, and within each subject as a function of distance from the
 fovea, remaining images were spatially normalized to a common template. At
 each distance from the fovea, we sampled signal at a specific number of
 points spaced evenly between the aforementioned anatomical borders: (a) 8
 points between the vitreous/RNFL and the RNFL/GCL borders, (b) 22 points
 between the RNFL/GCL border and the IPL/INL border, (c) 16 points between
 the IPL/INL border and the OPL/HFL border, (d) 16 points between the
 OPL/HFL border and the OLM, and (e) 18 points between the OLM and the RPE.
 Note that where a layer is thick (e.g., RNFL near the optic disc), those
 points are spaced farther apart than where the layer in thin (e.g., RNFL
 near the fovea). By dynamically over- or under-sampling based on layer
 thickness, a universal map of signal intensities as a function of location
 is generated. This map is available in ANALYZE format with the
 "_flat-normed_" prefix. Finally, this map is averaged to create
 a profile of reflectivity as a function of %Depth into the retina for each
 image (with the retina-vitreous border at 0%Depth, and the RPE at
 100%Depth, and intervals of 1.25%Depth). For completeness, five points are
 evenly sampled between 4 μm and 12 μm interior to retina (into the
 vitreous) and 4 μm and 24 μm exterior to the RPE (into the
 choriocapillaris) and placed at 1.25% intervals on this %Depth scale.
 These profiles are stored in .xlsx files in each folder.
   Profile data are also stored in PROFILE_DATA_for_Dryad.xlsx,
 alongside participant age ranges and other data. Statistical comparisons
 were performed by loading the tab-delimated version of the table
 (PROFILE_DATA_for_Dryad.txt) into R. These analyses scripts are not
 included in this repository, but are available upon request. In those
 final steps of the analysis, reflectivities were normalized: In each
 profile of reflectivity as a function of %Depth into the retina,
 reflectivites were ratio-normalized to the RPE (i.e., each value was
 divided by the reflectivity at 100%Depth, and multiplied by 100). It is
 also within these final R scripts that we were able to use the apparent
 angle of the retina as a covariate, accounting for the layer-specific
 impact of beam angle on reflectivity.  
description:   Missing and Excluded Data FOR EXPERIMENT 1: No subjects had
 gross signs of retinal pathology on infrared fundus images, nor on OCT
 images. Images were individually inspected to verify proper acquisition,
 adequate signal-to-noise, and the absence of imaging artifacts. In total,
 16 of 320 images were excluded. Each subject had at least 12 “bright”
 images in both lights “on” and lights “off” conditions, and at least 4
 “dim” images in both “on” and “off” conditions.  
 Missing and Excluded Data FOR EXPERIMENT 2: In total, 9 of 60 images were
 excluded (five “off” and four “on” images).   Missing
 and Excluded Data FOR EXPERIMENT 3: Only one image was excluded, an “on”
 image from the third patient. The first patient had extensive degeneration
 of the GCL, and data assigned to the GCL area by the spatial normalization
 program were therefore censored.   Missing and Excluded
 Data FOR EXPERIMENT 4: One Alzheimer’s patient had a sub-RPE deposit
 (presumably drusen) near the fovea, but the rest of the retinal strip had
 a benign appearance, visual fields were full, and Snellen acuity was
 20/30-2. Reflectivity profiles (in the subject-specific .xlsx file, as
 well as PROFILE_DATA_for_Dryad.xlsx) were calculated using the span from
 1000 to 2750 μm from the fovea, bypassing the deposit. Although a
 different participant had an abnormally thin RPE directly at the fovea
 (within 150 μm of its center), all data were retained due to the otherwise
 benign appearance of the retina, normal visual fields, and 20/30-2 Snellen
 acuity. Again, all images were reviewed for quality, and only five were
 excluded (each in a different participant), leaving 275 images.
   Exclusions due to quality or artifact (where present) are
 listed in a plain .txt file in each participant's folder.