Automated image processing pipeline for adaptive optics scanning light ophthalmoscopy.

To mitigate the substantial post-processing burden associated with adaptive optics scanning light ophthalmoscopy (AOSLO), we have developed an open-source, automated AOSLO image processing pipeline with both "live" and "full" modes. The live mode provides feedback during acquisition, while the full mode is intended to automatically integrate the copious disparate modules currently used in generating analyzable montages. The mean (±SD) lag between initiation and montage placement for the live pipeline was 54.6 ± 32.7s. The full pipeline reduced overall human operator time by 54.9 ± 28.4%, with no significant difference in resultant cone density metrics. The reduced overhead decreases both the technical burden and operating cost of AOSLO imaging, increasing overall clinical accessibility.

Optical Coherence Tomography Angiography in the Thirteen-Lined Ground Squirrel.

Purpose: To assess the performance of two spectral-domain optical coherence tomography-angiography systems in a natural model of hypoperfusion: the hibernating thirteen-lined ground squirrel (13-LGS). Methods: Using a high-speed (130 kHz) OCT-A system (HS-OCT-A) and a commercial OCT (36 kHz; Bioptigen Envisu; BE-OCT-A), we imaged the 13-LGS retina throughout its hibernation cycle. Custom software was used to extract the superior, middle, and deep capillary plexus (SCP, MCP, and DCP, respectively). The retinal vasculature was also imaged with adaptive optics scanning light ophthalmoscopy (AOSLO) during torpor to visualize individual blood cells. Finally, correlative histology with immunolabeled or DiI-stained vasculature was performed. Results: During euthermia, vessel density was similar between devices for the SCP and MCP (P = 0.88, 0.72, respectively), with a small difference in the DCP (-1.63 ± 1.54%, P = 0.036). Apparent capillary dropout was observed during torpor, but recovered after forced arousal, and this effect was exaggerated in high-speed OCT-A imaging. Based on cell flux measurements with AOSLO, increasing OCT-A scan duration by ∼1000× would avoid the apparent capillary dropout artifact. High correspondence between OCT-A (during euthermia) and histology enabled lateral scale calibration. Conclusions: While the HS-OCT-A system provides a more efficient workflow, the shorter interscan interval may render it more susceptible to the apparent capillary dropout artifact. Disambiguation between capillary dropout and transient ischemia can have important implications in the management of retinal disease and warrants additional diagnostics. Translational Relevance: The 13-LGS provides a natural model of hypoperfusion that may prove valuable in modeling the utility of OCT-A in human pathologies associated with altered blood flow.

Cone photoreceptor reflectance variation in the northern tree shrew and thirteen-lined ground squirrel.

In vivo images of human cone photoreceptors have been shown to vary in their reflectance both spatially and temporally. While it is generally accepted that the unique anatomy and physiology of the photoreceptors themselves drives this behavior, the exact mechanisms have not been fully elucidated as most studies on these phenomena have been limited to the human retina. Unlike humans, animal models offer the ability to experimentally manipulate the retina and perform direct in vivo and ex vivo comparisons. The thirteen-lined ground squirrel and northern tree shrew are two emerging animal models being used in vision research. Both models feature cone-dominant retinas, overcoming a key limitation of traditional rodent models. Additionally, each possesses unique but well-documented anatomical differences in cone structure compared to human cones, which can be leveraged to further constrain theoretical models of light propagation within photoreceptors. Here we sought to characterize the spatial and temporal reflectance behavior of cones in these species. Adaptive optics scanning light ophthalmoscopy (AOSLO) was used to non-invasively image the photoreceptors of both species at 5 to 10 min intervals over the span of 18 to 25 min. The reflectance of individual cone photoreceptors was measured over time, and images at individual time points were used to assess the variability of cone reflectance across the cone mosaic. Variability in spatial and temporal photoreceptor reflectance was observed in both species, with similar behavior to that seen in human AOSLO images. Despite the unique cone structure in these animals, these data suggest a common origin of photoreceptor reflectance behavior across species. Such data may help constrain models of the cellular origins of photoreceptor reflectance signals. These animal models provide an experimental platform to further explore the morphological origins of light capture and propagation.

Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins.

Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.

Visual Acuity and Foveal Structure in Eyes with Fragmented Foveal Avascular Zones.

PURPOSE: To assess the frequency and impact of abnormal foveal avascular zone (FAZ) topography (i.e., a fragmented FAZ) on visual acuity and foveal anatomic features. DESIGN: Prospective, cross-sectional study from March 2018 through July 2019. PARTICIPANTS: Two-hundred fifty participants were screened from a normative OCT angiography database. Of those, 12 participants were found to have at least 1 eye with a fragmented FAZ. Eight returned for follow-up imaging, along with an additional 3 participants with ocular disease (amblyopia, autosomal recessive bestrophinopathy, premature birth) having a similar FAZ phenotype. METHODS: Follow-up OCT imaging and monocular best-corrected visual acuity (BCVA) were performed for these 11 participants. Twenty-four participants with a clearly defined FAZ were recruited for comparison. A normative database was created measuring parafoveal intercapillary area (PICA) to determine if an FAZ was fragmented. MAIN OUTCOME MEASURES: Monocular BCVA, foveal pit depth, foveal pit area, PICA, outer nuclear layer thickness, foveal inner retinal area, and peak cone density. RESULTS: The frequency of a fragmented FAZ was 4.8% of individuals (12 of 250) or 3.6% of eyes (18 of 500 eyes). A significant difference was found between the control eyes and eyes with fragmented FAZs for foveal pit depth, pit area, and total PICA (P < 0.001, P = 0.002, and P < 0.001, respectively). The presence of a fragmented FAZ did not affect visual acuity. CONCLUSIONS: The presence of a fragmented FAZ seems not to be a rare phenotype in individuals with normal vision. The presence of altered FAZ topography in patients with retinal or systemic disease could negatively impact the accuracy and sensitivity of biomarkers dependent on FAZ identification.

Noninvasive Imaging and Correlative Histology of Cone Photoreceptor Structure in the Pig Retina.

PURPOSE: To evaluate different methods of studying cone photoreceptor structure in wild-type (WT) and transgenic pigs carrying the human rhodopsin P23H mutant gene (TgP23H). METHODS: For in vivo imaging, pigs were anesthetized with tiletamine-zolazepam and isoflurane and given lidocaine-bupivacaine retrobulbar injections. Stay sutures and a custom head mount were used to hold and steer the head for adaptive optics scanning light ophthalmoscopy (AOSLO). Six WT and TgP23H littermates were imaged at postnatal day 30 (P30), P90, and P180 with AOSLO and optical coherence tomography (OCT), and two additional sets of littermates were imaged at P3 and P15 with OCT only. AOSLO imaging and correlative differential interference contrast microscopy were performed on a P240 WT pig and on WT and TgP23H littermates at P30 and P180. RESULTS: AOSLO cone density generally underestimates histology density (mean difference ± SD = 24.8% ± 21.4%). The intensity of the outer retinal hyperreflective OCT band attributed to photoreceptors is attenuated in TgP23H pigs at all ages. In contrast, AOSLO images show cones that retain inner and outer segments through P180. At retinal locations outside the visual streak, TgP23H pigs show a heterogeneous degenerating cone mosaic by using two criteria: variable contrast on a split detector AOSLO and high reflectivity on a confocal AOSLO. CONCLUSIONS: AOSLO reveals that the cone mosaic is similar to ex vivo histology. Its use as a noninvasive tool will enable observation of morphologic changes that arise in the cone mosaic of TgP23H pigs over time. TRANSLATIONAL RELEVANCE: Pigs are widely used for translational studies, and the ability to noninvasively assess cellular changes in the cone mosaic will facilitate more detailed investigations of new retinal disease models as well as outcomes of potential therapies.

Interocular symmetry, intraobserver repeatability, and interobserver reliability of cone density measurements in the 13-lined ground squirrel.

BACKGROUND: The 13-lined ground squirrel (13-LGS) possesses a cone-dominant retina that is highly amenable to non-invasive high-resolution retinal imaging. The ability for longitudinal assessment of a cone-dominant photoreceptor mosaic with an adaptive optics scanning light ophthalmoscope (AOSLO) has positioned the 13-LGS to become an accessible model for vision research. Here, we examine the interocular symmetry, repeatability, and reliability of cone density measurements in the 13-LGS. METHODS: Thirteen 13-LGS (18 eyes) were imaged along the vertical meridian with a custom AOSLO. Regions of interest were selected superior and inferior to the optic nerve head, including the cone-rich visual streak. Non-confocal split-detection was used to capture images of the cone mosaic. Five masked observers each manually identified photoreceptors for 26 images three times and corrected an algorithm's cell identification outputs for all 214 images three times. Intraobserver repeatability and interobserver reliability of cone density were characterized using data collected from all five observers, while interocular symmetry was assessed in five animals using the average values of all observers. The distribution of image quality for all images in this study was assessed with open-sourced software. RESULTS: Manual identification was less repeatable than semi-automated correction for four of the five observers. Excellent repeatability was seen from all observers (ICC = 0.997-0.999), and there was good agreement between repeat cell identification corrections in all five observers (range: 9.43-25.71 cells/degree2). Reliability of cell identification was significantly different in two of the five observers, and worst in images taken from hibernating 13-LGS. Interocular symmetry of cone density was seen in the five 13-LGS assessed. Image quality was variable between blur- and pixel intensity-based metrics. CONCLUSIONS: Interocular symmetry with repeatable cone density measurements suggest that the 13-LGS is well-suited for longitudinal examination of the cone mosaic using split-detection AOSLO. Differences in reliability highlight the importance of observer training and automation of AOSLO cell detection. Cone density measurements from hibernating 13-LGS are not repeatable. Additional studies are warranted to assess other metrics of cone health to detect deviations from normal 13-LGS in future models of cone disorder in this species.

Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology.

Tree shrews are small mammals with excellent vision and are closely related to primates. They have been used extensively as a model for studying refractive development, myopia, and central visual processing and are becoming an important model for vision research. Their cone dominant retina (∼95% cones) provides a potential avenue to create new damage/disease models of human macular pathology and to monitor progression or treatment response. To continue the development of the tree shrew as an animal model, we provide here the first measurements of higher order aberrations along with adaptive optics scanning light ophthalmoscopy (AOSLO) images of the photoreceptor mosaic in the tree shrew retina. To compare intra-animal in vivo and ex vivo cone density measurements, the AOSLO images were matched to whole-mount immunofluorescence microscopy. Analysis of the tree shrew wavefront indicated that the optics are well-matched to the sampling of the cone mosaic and is consistent with the suggestion that juvenile tree shrews are nearly emmetropic (slightly hyperopic). Compared with in vivo measurements, consistently higher cone density was measured ex vivo, likely due to tissue shrinkage during histological processing. Tree shrews also possess massive mitochondria ("megamitochondria") in their cone inner segments, providing a natural model to assess how mitochondrial size affects in vivo retinal imagery. Intra-animal in vivo and ex vivo axial distance measurements were made in the outer retina with optical coherence tomography (OCT) and transmission electron microscopy (TEM), respectively, to determine the origin of sub-cellular cone reflectivity seen on OCT. These results demonstrate that these megamitochondria create an additional hyper-reflective outer retinal reflective band in OCT images. The ability to use noninvasive retinal imaging in tree shrews supports development of this species as a model of cone disorders.

Evaluating seasonal changes of cone photoreceptor structure in the 13-lined ground squirrel.

Cone photoreceptors of the 13-lined ground squirrel (13-LGS) undergo reversible structural changes during hibernation, including cone outer segment disc degeneration and inner segment mitochondria depletion. Here, we evaluated cone structure with adaptive optics scanning light ophthalmoscopy (AOSLO) before, during, and after hibernation. Also, intra-animal comparisons of cone structure were made at distinct physiological states (pre-hibernation, torpor, interbout euthermia, and post-hibernation) with AOSLO and transmission electron microscopy. Our results indicate that the 13-LGS cone mosaic is only transiently affected by structural remodeling during hibernation. Outer segment remodeling starts during torpid states during a period of fall transition in room temperature, with more severe structural changes during bouts of torpor in cold temperature. Cones return to euthermic-like structure during brief periods of interbout euthermia and recover normal waveguiding properties as soon as 24 h post-hibernation. Cone structure is visible with split-detector AOSLO throughout hibernation, providing evidence that intact outer segments are not necessary to visualize cones with this technique. Despite the changes to cone structure during hibernation, cone density and packing remained unchanged throughout the seasonal cycle. Pairing non-invasive imaging with ultrastructural assessment may provide insight to the biological origins of cone photoreceptor signals observed with AOSLO.

Acid Ceramidase Deficiency in Mice Leads to Severe Ocular Pathology and Visual Impairment.

Farber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment.

Assessing the Accuracy of Foveal Avascular Zone Measurements Using Optical Coherence Tomography Angiography: Segmentation and Scaling.

PURPOSE: The foveal avascular zone (FAZ) is altered in numerous diseases. We assessed factors (axial length, segmentation method, age, sex) impacting FAZ measurements from optical coherence tomography (OCT) angiography images. METHODS: We recruited 116 Caucasian subjects without ocular disease, and acquired two 3 × 3 mm AngioVue scans per each right eye (232 total scans). In images of the superficial plexus, the FAZ was segmented using the AngioVue semiautomatic nonflow measurement tool and ImageJ manual segmentation. In images from the full retinal thickness, the FAZ was segmented using the AngioAnalytics automatic FAZ tool. Repeatability, reliability, and reproducibility were calculated for FAZ measurements (acircularity, area). RESULTS: FAZ area (mean ± SD) for manual segmentation was 0.240 ± 0.0965 mm2, greater than both semiautomatic (0.216 ± 0.0873 mm2) and automatic (0.218 ± 0.0869 mm2) segmentation (P < 0.05). Not correcting for axial length introduced errors up to 25% in FAZ area. Manual area segmentation had better repeatability (0.020 mm2) than semiautomatic (0.043 mm2) or automatic (0.056 mm2). FAZ acircularity had better repeatability with automatic than manual segmentation (0.086 vs. 0.114). Reliability of all area measurements was excellent (intraclass correlation coefficient [ICC] = 0.994 manual, 0.969 semiautomatic, 0.948 automatic). Reliability of acircularity measurements was 0.879 for manual and 0.606 for automatic. CONCLUSION: We identified numerous factors affecting FAZ measurements. These errors confound comparisons across studies and studies examining factors that may correlate with FAZ measures. TRANSLATIONAL RELEVANCE: Using FAZ measurements as biomarkers for disease progression requires assessing and controlling for different sources of error. Not correcting for ocular magnification can result in significant inaccuracy in FAZ measurements, while choice of segmentation method affects both repeatability and accuracy.

An Automated Reference Frame Selection (ARFS) Algorithm for Cone Imaging with Adaptive Optics Scanning Light Ophthalmoscopy.

PURPOSE: To develop an automated reference frame selection (ARFS) algorithm to replace the subjective approach of manually selecting reference frames for processing adaptive optics scanning light ophthalmoscope (AOSLO) videos of cone photoreceptors. METHODS: Relative distortion was measured within individual frames before conducting image-based motion tracking and sorting of frames into distinct spatial clusters. AOSLO images from nine healthy subjects were processed using ARFS and human-derived reference frames, then aligned to undistorted AO-flood images by nonlinear registration and the registration transformations were compared. The frequency at which humans selected reference frames that were rejected by ARFS was calculated in 35 datasets from healthy subjects, and subjects with achromatopsia, albinism, or retinitis pigmentosa. The level of distortion in this set of human-derived reference frames was assessed. RESULTS: The average transformation vector magnitude required for registration of AOSLO images to AO-flood images was significantly reduced from 3.33 ± 1.61 pixels when using manual reference frame selection to 2.75 ± 1.60 pixels (mean ± SD) when using ARFS (P = 0.0016). Between 5.16% and 39.22% of human-derived frames were rejected by ARFS. Only 2.71% to 7.73% of human-derived frames were ranked in the top 5% of least distorted frames. CONCLUSION: ARFS outperforms expert observers in selecting minimally distorted reference frames in AOSLO image sequences. The low success rate in human frame choice illustrates the difficulty in subjectively assessing image distortion. TRANSLATIONAL RELEVANCE: Manual reference frame selection represented a significant barrier to a fully automated image-processing pipeline (including montaging, cone identification, and metric extraction). The approach presented here will aid in the clinical translation of AOSLO imaging.