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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 (â¼95% 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.
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Aberrações de Frente de Onda da Córnea/fisiopatologia , Erros de Refração/fisiopatologia , Retina/diagnóstico por imagem , Células Fotorreceptoras Retinianas Cones/citologia , Aberrometria , Animais , Contagem de Células , Microscopia Eletrônica de Transmissão , Oftalmoscopia , Imagem Óptica , Refração Ocular/fisiologia , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Tomografia de Coerência Óptica/métodos , TupaiaRESUMO
Quantification of the rod photoreceptor mosaic using adaptive optics scanning light ophthalmoscopy (AOSLO) remains challenging. Here we demonstrate a method for deriving estimates of rod density and rod:cone ratio based on measures of rod spacing, cone numerosity, and cone inner segment area. Twenty-two AOSLO images with complete rod visualization were used to validate this spacing-derived method for estimating density. The method was then used to estimate rod metrics in an additional 105 images without complete rod visualization. The spacing-derived rod mosaic metrics were comparable to published data from histology. This method could be leveraged to develop large normative databases of rod mosaic metrics, though limitations persist with intergrader variability in assessing cone area and numerosity.
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PURPOSE: To describe cone structure changes using adaptive optics scanning laser ophthalmoscopy (AOSLO) in the Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A) study. DESIGN: Multicenter, longitudinal natural history study. METHODS: AOSLO images were acquired at 4 centers, twice at baseline and annually for 24 months in this natural history study. For each eye, at least 10 regions of interest (ROIs) with ≥50 contiguous cones were analyzed by masked, independent graders. Cone spacing Z-scores, standard deviations from the normal mean at the measured location, were compared between graders and tests at baseline. The association of cone spacing with clinical characteristics was assessed using linear mixed effects regression models weighted by image quality score. Annual rates of change were calculated based on differences between visits. RESULTS: Fourteen eyes of 14 participants were imaged, with 192 ROIs selected at baseline. There was variability among graders, which was greater in images with lower image quality score (P < .001). Cone spacing was significantly correlated with eccentricity, quality score, and disease duration (P < .02). On average, the cone spacing Z-score increased 0.14 annually (about 9%, P < .001). We observed no significant differences in rate of change between disease type (Usher syndrome or retinitis pigmentosa), imaging site, or grader. CONCLUSIONS: Using current methods, the analysis of quantitative measures of cone structure showed some challenges, yet showed promise that AOSLO images can be used to characterize progressive change over 24 months. Additional multicenter studies using AOSLO are needed to advance cone mosaic metrics as sensitive outcome measures for clinical trials. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.
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Degeneração Retiniana , Síndromes de Usher , Humanos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/genética , Tomografia de Coerência Óptica/métodos , Células Fotorreceptoras Retinianas Cones , Oftalmoscopia/métodos , Proteínas da Matriz ExtracelularRESUMO
The foveal cone mosaic can be directly visualized using adaptive optics scanning light ophthalmoscopy (AOSLO). Previous studies in individuals with normal vision report wide variability in the topography of the foveal cone mosaic, especially the value of peak cone density (PCD). While these studies often involve a human grader, there have been no studies examining intergrader reproducibility of foveal cone mosaic metrics. Here we re-analyzed published AOSLO foveal cone images from 44 individuals to assess the relationship between the cone density centroid (CDC) location and the location of PCD. Across 5 graders with variable experience, we found a measurement error of 11.7% in PCD estimates and higher intergrader reproducibility of CDC location compared to PCD location (p < 0.0001). These estimates of measurement error can be used in future studies of the foveal cone mosaic, and our results support use of the CDC location as a more reproducible anchor for cross-modality analyses.
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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.
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PURPOSE: Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging. METHODS: One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL). RESULTS: Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects. CONCLUSIONS: This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.
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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.
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Oftalmoscopia/métodos , Retina/anatomia & histologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sciuridae/anatomia & histologia , Tupaia/anatomia & histologia , Animais , Feminino , Masculino , Modelos Animais , Fatores de TempoRESUMO
PURPOSE: To determine the axial location of Gunn's dots in the retina. METHODS: Adaptive optics scanning laser ophthalmoscopy (AOSLO) images and adaptive optics - optical coherence tomography (AO-OCT) volumes were collected from a region where Gunn's dots were found inferior to the optic disc from a subject determined by clinical examination to be a glaucoma suspect. AO-OCT volumes were also collected along the horizontal and vertical meridians from six healthy subjects and one glaucoma subject to identify and document other occurrences of Gunn's dots. AO-OCT volumes were registered in three-dimensions and averaged. Gunn's dots were segmented, and their volume, area, and diameter were measured. RESULTS: All Gunn's dots imaged in this study from all subjects were confined to the inner limiting membrane, neither extending into the vitreous nor into the nerve fiber layer. The size of the dots was highly variable. The measured volume, area, and diameter (mean ± standard deviation) were 1119.9 ± 590.9 µm3, 220.2 ± 105.5 µm2, and 14.3 ± 3.1 µm, the latter within the range as previously published reports. CONCLUSIONS: Based upon evidence from this study and others, Gunn's dots are not thought to be Müller cell end-feet or hyalocytes. We hypothesize that they are related to microglia, either as the by-product of their phagocytosis function, or are actual dead ameboid-shaped microglia who have fulfilled their scavenger role in retinal pathology. Further studies are needed in diseased eyes to determine if they have predictive value.
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Purpose: To test the hypothesis that foveal cone topography is symmetrical between contralateral eyes. Methods: We used adaptive optics scanning light ophthalmoscopy to acquire images of the foveal cone mosaic in each eye of 58 subjects with normal vision (35 female, 23 male). Cones were semiautomatically identified over a 300 × 300-µm foveal area. From these cone coordinates, maps of cone density were derived, and we extracted estimates of peak cone density from each map. Mosaic regularity was assessed using Voronoi cell area regularity (VCAR). Average roundness and average area of the 70%, 75%, 80%, 85%, and 90% of peak density isodensity contours were evaluated. Results: The average peak cone density for right eyes was 180,286 cones/mm2 (n = 49) and for left eyes was 182,397 cones/mm2 (n = 45), with a mean absolute difference of 6363 cones/mm2 (n = 43). Peak density, cone spacing, VCAR, and average area within the isodensity contours of fellow eyes were not significantly different (P = 0.60, P = 0.83, P = 0.30, and P = 0.39, respectively). However, the average roundness of the isodensity contours was 2% more circular in the right eyes than in the left eyes (P = 0.02). Conclusions: There is interocular symmetry of peak foveal cone density, mosaic regularity, and area encompassing the most densely packed cells in subjects with normal vision. The origin and significance of the observed interocular difference in average roundness of the isodensity contours are unclear.
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Fóvea Central/anatomia & histologia , Células Fotorreceptoras Retinianas Cones/citologia , Adolescente , Adulto , Idoso , Contagem de Células , Criança , Feminino , Fóvea Central/citologia , Fóvea Central/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Oftalmoscopia , Adulto JovemRESUMO
Purpose: To examine the foveal avascular zone (FAZ) in patients with congenital achromatopsia (ACHM). Methods: Forty-two patients with genetically confirmed ACHM were imaged either with Optovue's AngioVue system or Zeiss's Plex Elite 9000, and the presence or absence of a FAZ was determined. For images where a FAZ was present and could be confidently segmented, FAZ area, circularity index, and roundness were measured and compared with previously published normative values. Structural optical coherence tomography images were acquired to assess the degree of foveal hypoplasia (number and thickness of inner retinal layers present at the fovea). Results: A FAZ was present in 31 of 42 patients imaged (74%), although no determination could be made for 11 patients due to poor image quality (26%). The mean ± SD FAZ area for the ACHM retina was 0.281 ± 0.112 mm2, which was not significantly different from the previously published normative values (P = 0.94). However, their FAZs had decreased circularity (P < 0.0001) and decreased roundness (P < 0.0001) compared to the normative cohort. In the patients with ACHM examined here, the FAZ area decreased as the number and thickness of the retained inner retinal layers increased. Conclusions: Our data demonstrate that despite the presence of foveal hypoplasia, patients with ACHM can have a FAZ. This is distinct from other conditions associated with foveal hypoplasia, which generally show an absence of the FAZ. In ACHM, FAZ formation does not appear to be sufficient for complete pit formation, contrary to some models of foveal development.
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Defeitos da Visão Cromática/congênito , Fóvea Central/patologia , Adolescente , Adulto , Idoso , Criança , Defeitos da Visão Cromática/diagnóstico por imagem , Defeitos da Visão Cromática/patologia , Feminino , Fóvea Central/irrigação sanguínea , Fóvea Central/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Retina/diagnóstico por imagem , Retina/patologia , Vasos Retinianos/diagnóstico por imagem , Vasos Retinianos/patologia , Tomografia de Coerência Óptica , Adulto JovemRESUMO
Purpose: The purpose of this study was to investigate the effect of device and scan size on quantitative optical coherence tomography angiography (OCT-A) metrics. Methods: The 3 × 3 mm scans from Optovue AngioVue and Zeiss AngioPlex systems were included for 18 eyes of 18 subjects without ocular pathology. The foveal avascular zone (FAZ) was segmented manually by two observers, from which estimates of FAZ area (using both the nominal image scale and the axial length corrected image scale) and acircularity were derived. Three scan sizes (3 mm, 6 mm HD, and 8 mm) from the AngioVue system were included for 15 eyes of 15 subjects without ocular pathology. For each subject, larger image sizes were resized to the same resolution as 3 × 3 mm scans, aligned, then cropped to a common area. FAZ area, FAZ acircularity, average and total parafoveal intercapillary area, vessel density, and vessel end points were computed. Results: Between the devices used here, there were no significant differences in FAZ acircularity (P = 0.88) or FAZ area using scaled (P = 0.11) or unscaled images (P = 0.069). Although there was no significant difference in FAZ area across scan sizes (P = 0.30), vessel morphometry metrics were all significantly influenced by scan size. Conclusions: The scan devices and sizes used here do not affect FAZ area measures derived from manual segmentations. In contrast, vessel morphometry metrics are affected by scan size. As individual differences in axial length induce differences in absolute scan size, extreme care should be taken when interpreting metrics of vessel morphometry, both between and within OCT-A devices. Translational Relevance: A better characterization of the confounds surrounding OCT-A retinal vasculature metrics can lead to improved application of these metrics as biomarkers for retinal and systemic diseases.
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Benchmarking , Tomografia de Coerência Óptica , Angiofluoresceinografia , Fóvea Central , Humanos , Vasos Retinianos/diagnóstico por imagemRESUMO
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.
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Angiofluoresceinografia/métodos , Fóvea Central/irrigação sanguínea , Vasos Retinianos/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Acuidade Visual , Adolescente , Adulto , Criança , Estudos Transversais , Feminino , Fundo de Olho , Humanos , Masculino , Estudos Prospectivos , Adulto JovemRESUMO
Heritability and evolvability estimates of adult traits from free-living bird populations can be used to gauge the ability of populations to respond to selection, but are rare due to difficulties in gathering detailed pedigree information. The capacity to respond to selection is particularly important for species occupying managed habitats such as agricultural grasslands because of the potential for humans to accidentally influence traits. We calculated heritability and evolvability of six morphological traits in a population of Savannah Sparrows (Passerculus sandwichensis) breeding in a large agricultural landscape. We used microsatellite analysis to determine a genetic pedigree, revealing a high level of extra-pair paternity (63%) within a relatively philopatric population. For the entire population, heritabilities varied from low to high (bill width: 0.160±0.182 to tarsus length: 0.651±0.155), while evolvabilities were low across all traits (wing length: 0.035±0.013 to body mass: 0.066±0.106). Our results indicate that any directional selection from agricultural management practices will produce negligible changes in basic morphometrics of Savannah sparrow populations occupying the Champlain Valley of Vermont, USA.
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Agricultura , Evolução Biológica , Cruzamento , Pradaria , Padrões de Herança/genética , Pardais/genética , Animais , Loci Gênicos , Repetições de Microssatélites/genética , Fenótipo , Característica Quantitativa Herdável , TemperaturaRESUMO
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.
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Oftalmoscopia/métodos , Células Fotorreceptoras Retinianas Cones/fisiologia , Sciuridae/fisiologia , Animais , Feminino , Masculino , Modelos Animais , Oftalmoscópios , Reprodutibilidade dos TestesRESUMO
Offspring sex ratios at the termination of parental care should theoretically be skewed toward the less expensive sex, which in most avian species would be females, the smaller gender. Among birds, however, raptors offer an unusual dynamic because they exhibit reversed size dimorphism with females being larger than males. And thus theory would predict a preponderance of male offspring. Results for raptors and birds in general have been varied although population-level estimates of sex ratios in avian offspring are generally at unity. Adaptive adjustment of sex ratios in avian offspring is difficult to predict perhaps in part due to a lack of life-history details and short-term investigations that cannot account for precision or repeatability of sex ratios across time. We conducted a novel comparative study of sex ratios in nestling Cooper's hawks (Accipiter cooperii) in two study populations across breeding generations during 11 years in Wisconsin, 2001-2011. One breeding population recently colonized metropolitan Milwaukee and exhibited rapidly increasing population growth, while the ex-Milwaukee breeding population was stable. Following life-history trade-off theory and our prediction regarding this socially monogamous species in which reversed sexual size dimorphism is extreme, first-time breeding one-year-old, second-year females in both study populations produced a preponderance of the smaller and cheaper sex, males, whereas ASY (after-second-year), ≥2-year-old females in Milwaukee produced a nestling sex ratio near unity and predictably therefore a greater proportion of females compared to ASY females in ex-Milwaukee who produced a preponderance of males. Adjustment of sex ratios in both study populations occurred at conception. Life histories and selective pressures related to breeding population trajectory in two age cohorts of nesting female Cooper's hawk likely vary, and it is possible that these differences influenced the sex ratios we documented for two age cohorts of female Cooper's hawks in Wisconsin.