Bruch's Membrane Calcification in Pseudoxanthoma Elasticum: Comparing Histopathology and Clinical Imaging.

Purpose: To investigate the histology of Bruch's membrane (BM) calcification in pseudoxanthoma elasticum (PXE) and correlate this to clinical retinal imaging. Design: Experimental study with clinicopathological correlation. Subjects and Controls: Six postmortem eyes from 4 PXE patients and 1 comparison eye from an anonymous donor without PXE. One of the eyes had a multimodal clinical image set for comparison. Methods: Calcification was labeled with OsteSense 680RD, a fluorescent dye specific for hydroxyapatite, and visualized with confocal microscopy. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy (SEM-EDX) and time-of-flight secondary ion mass spectrometry (TOF-SIMs) were used to analyze the elemental and ionic composition of different anatomical locations. Findings on cadaver tissues were compared with clinical imaging of 1 PXE patient. Main Outcome Measures: The characteristics and topographical distribution of hydroxyapatite in BM in eyes with PXE were compared with the clinical manifestations of the disease. Results: Analyses of whole-mount and sectioned PXE eyes revealed an extensive, confluent OsteoSense labeling in the central and midperipheral BM, transitioning to a speckled labeling in the midperiphery. These areas corresponded to hyperreflective and isoreflective zones on clinical imaging. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy and TOF-SIMs analyses identified these calcifications as hydroxyapatite in BM of PXE eyes. The confluent fluorescent appearance originates from heavily calcified fibrous structures of both the collagen and the elastic layers of BM. Calcification was also detected in an aged comparison eye, but this was markedly different from PXE eyes and presented as small snowflake-like deposits in the posterior pole. Conclusions: Pseudoxanthoma elasticum eyes show extensive hydroxyapatite deposition in the inner and outer collagenous and elastic BM layers in the macula with a gradual change toward the midperiphery, which seems to correlate with the clinical phenotype. The snowflake-like calcification in BM of an aged comparison eye differed markedly from the extensive calcification in PXE. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The Double Layer Sign Is Highly Predictive of Progression to Exudation in Age-Related Macular Degeneration.

PURPOSE: The presences of a double layer sign (DLS) and a shallow irregular retinal pigment epithelium (RPE) elevation (SIRE) were investigated using spectral domain-OCT (SD-OCT) imaging to determine their ability to predict progression to exudative macular neovascularization (eMNV) in the unaffected fellow eyes (study eye) of participants with age-related macular degeneration (AMD) with newly diagnosed unilateral eMNV. DESIGN: Retrospective, reanalysis of SD-OCT scans of study eyes from the Early Detection of Neovascular AMD (EDNA) study with 3 years follow-up (FU). PARTICIPANTS: The EDNA study repository of SD-OCT scans was assessed for inclusion. Cases with incomplete data sets, low quality scans, or exhibiting other pathology were excluded, which resulted in 459 eligible cases. METHODS: Spectral domain-OCT volume scans of study eyes were graded for irregular elevation of the RPE (IE), with length, and height measurements made on the most affected B-scan. Eyes with heterogeneous reflectivity within the IE were classified as exhibiting the DLS. Eyes with DLS where the length of separation between RPE and Bruch's membrane was ≥ 1000 µm in length and < 100 µm in height were subclassified as SIRE. MAIN OUTCOME MEASURES: Hazard of progression to eMNV for DLS and SIRE. RESULTS: Of the 459 eyes, 268 had IE, of which 101 were DLS-like and 51 also fulfilled criteria for SIRE. Over the 3 years FU period, 104 (23%) eyes progressed to eMNV. After an FU of 18 months, a significantly higher proportion of study eyes (P < 0.001) with IE, DLS, and SIRE developed eMNV compared with those without these features (IE: 17% vs. no IE 6.3%; DLS: 23% vs. no DLS 9.9%; SIRE: 22% vs. no SIRE 11%). In the adjusted Cox regression models, a significantly greater hazard of progression (P < 0.001) was associated with the presence of IE (adjusted hazard ratio [HR], 3.01; 95% confidence interval [CI], 1.88-4.82), DLS (adjusted HR, 3.41; 95% CI, 2.26-5.14), or SIRE (adjusted HR, 2.83; 95% CI, 1.68-4.75). CONCLUSION: The DLS is a highly sensitive predictor of progression to eMNV, and the use of SIRE does not improve predictability. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Reduced Cone Density Is Associated with Multiple Sclerosis.

Purpose: Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system. Recent evidence suggests that degeneration of the inner layers of the retina occurs in MS. This study aimed to examine whether there are outer retinal changes in patients living with MS. Design: This was a single center, cross-sectional study. Participants: Sixteen patients with MS and 25 controls (volunteers without diagnosed MS) were recruited for the study. Methods: We acquired volumetric spectral domain-OCT scans of the macula and a circular scan around the optic nerve head (ONH). We also captured adaptive optics (AO) images at 0° (centered on the foveola), 2°, 4°, and 6° temporal to the fovea. Main Outcome Measures: We calculated the thickness of the different retinal layers in the macula and around the ONH using the inbuilt software of the OCT. We evaluated changes in cone photoreceptors by calculating cone density and spacing by the inbuilt AO automatic segmentation algorithm with manual correction. We compared patients with and without optic neuritis and controls. Results: We found significant thinning of the inner retina and a thickening of the outer retina in the eye with a history of optic neuritis (eyes of patients with MS with a history of optic neuritis; mean difference [MD]: -11.13 ± 3.61 µm, P = 0.002 and MD: 2.86 ± 0.89 µm, P = 0.001; respectively). We did not observe changes in retinal layers without optic neuritis in eyes of patients with MS without a history of optic neuritis. However, regional differences were detected in the peripapillary retinal nerve fiber layer. Analyzing AO images revealed a significantly lower cone outer-segment density at all eccentricities in all patients compared with control eyes (P < 0.05), independent of optic neuritis history. Conclusions: Our results showed that all MS cases were associated with decreased cone densities. Future longitudinal studies will help to elucidate whether this is a specific and sensitive method to detect and monitor the development and progression of MS. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Nuclear and cellular, micro and nano calcification in Alzheimer's disease patients and correlation to phosphorylated Tau.

Brain calcification (calcium phosphate mineral formation) has been reported in the past 100 years in the brains of Alzheimer's disease (AD) patients. However, the association between calcification and AD, the triggers for calcification, and its role within the disease are not clear. On the other hand, hyperphosphorylated Tau protein (pTau) tangles have been widely studied and recognized as an essential factor in developing AD. In this work, calcification in the brains of AD patients is characterized by advanced electron microscopy and fluorescence microscopy. Results are then compared to samples from cognitively healthy, age-matched donors, and the colocalization of calcification and pTau is investigated. Here, we show that AD patients' brains present microcalcification associated with the neural cell nuclei and cell projections, and that these are strongly related to the presence of pTau. The link between microcalcification and pTau suggests a potential mechanism of brain cell damage. Together with the formation of amyloid plaques and neurofibrillary tangles, microcalcification in neuronal cells adds to a better understanding of the pathology of AD. Finally, the presence of microcalcification in the neuronal cells of AD patients may assist in AD diagnosis, and may open avenues for developing intervention strategies based on inhibition of calcification. STATEMENT OF SIGNIFICANCE: Brain calcification has been reported in the past 100 years in the brains of Alzheimer's disease (AD) patients. However, the association between calcification and AD is not clear. Hyperphosphorylated Tau protein (pTau) has been studied and recognized as a key factor in developing AD. We show here that AD patients' brains present microcalcification associated with the neuronal cell nuclei and cell projections, and that these are related to the presence of pTau. The study of calcification in brain cells can contribute to a better understanding of the biochemical mechanisms associated with AD and might also reveal that calcification is part of the full disease mechanism. Moreover, this work opens the possibility for using calcification as a biomarker to identify AD.

Retinal phenotyping of variants of Alzheimer's disease using ultra-widefield retinal images.

BACKGROUND: Posterior cortical atrophy (PCA) is the most common atypical variant of Alzheimer's disease (AD). Changes associated with PCA in the brain affect the visual cortex, but little is known about retinal changes in PCA. In this study, we explored retinal phenotypic variations in typical AD (tAD) and PCA. METHODS: Retinal phenotyping was carried out on ultra-widefield (UWF) images of 69 control, 24 tAD, and 25 PCA participants. RESULTS: Individuals with tAD (odds ratio [OR] = 2.76 [confidence interval (CI):1.24 to 6.10], P = .012) and PCA (OR = 3.40 [CI:1.25 to 9.22], P = .016) were more likely phenotyped as hard drusen. tAD (OR = 0.34 [CI:0.12 to 0.92], P = .035) were less likely to have soft drusen compared to control. Almost 3-fold increase in reticular pseudodrusen formation in tAD (OR = 2.93 [CI:1.10 to 7.76], P = .030) compared to control was estimated. DISCUSSION: Studying the peripheral retina may contribute to a better understanding of differences in retinal phenotypes of different AD variants.

Increased choroidal thickness in adults with Down syndrome.

INTRODUCTION: People with Down syndrome (DS) are particularly susceptible to Alzheimer's disease (AD) due to the triplication of the amyloid precursor protein (APP) gene. In this cross-sectional study, we hypothesized that choroidal thinning reported in sporadic AD (sAD) is mirrored in adults with DS. METHODS: The posterior pole of the eye for 24 adults with DS and 16 age-matched controls (Ctrl) were imaged with optical coherence tomography. Choroidal thickness (ChT) was measured and analyzed in relation to cognitive status and cerebral amyloid beta (Aß) load. RESULTS: ChT was increased in people with DS (pwDS) compared to Ctrl. This increase was associated with gender differences and positively correlated with cerebral Aß load in a small subset. There was no significant correlation detected between ChT and age or cognitive status. DISCUSSION: In contrast to sAD this study found a significantly thicker choroid in pwDS. Whether these changes are related to Aß pathology in DS needs further investigation.

Characterization of Calcium Phosphate Spherical Particles in the Subretinal Pigment Epithelium-Basal Lamina Space in Aged Human Eyes.

Purpose: Micrometer-sized spherules formed of hydroxyapatite or whitlockite were identified within extracellular deposits that accumulate in the space between the basal lamina (BL) of retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch's membrane (sub-RPE-BL space). This investigation aimed to characterize the morphologic features, structure, and distribution of these spherules in aged human eyes with and without clinical indications of age-related macular degeneration (AMD). Design: Experimental study. Participants: Five human eyes with varying degrees of sub-RPE-BL deposits were obtained from the University College London Institute of Ophthalmology and Moorfield's Eye Hospital Tissue Repository or the Advancing Sight Network. Two eyes were reported as having clinical indications of AMD (age, 76-87 years), whereas 3 were considered healthy (age, 69-91 years). Methods: Cadaveric eyes with sub-RPE-BL deposits were embedded in paraffin wax and sectioned to a thickness of 4-10 µm. Spherules were identified and characterized using high-resolution scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy, and time-of-flight secondary ion mass spectroscopy. Main Outcome Measures: High-resolution scanning electron micrographs of spherules, the size-frequency distribution of spherules including average diameter, and the distribution of particles across the central-peripheral axis. Elemental maps and time-of-flight secondary ion mass spectra also were obtained. Results: The precipitation of spherules is ubiquitous across the central, mid-peripheral, and far-peripheral axis in aged human eyes. No significant difference was found in the frequency of spherules along this axis. However, statistical analysis indicated that spherules exhibited significantly different sizes in these regions. In-depth analysis revealed that spherules in the sub-RPE-BL space of eyes with clinical signs of AMD were significantly larger (median diameter, 1.64 µm) than those in healthy aged eyes (median diameter, 1.16 µm). Finally, spherules showed great variation in surface topography and internal structure. Conclusions: The precipitation of spherules in the sub-RPE-BL space is ubiquitous across the central-peripheral axis in aged human eyes. However, a marked difference exists in the size and frequency of spherules in eyes with clinical signs of AMD compared to those without, suggesting that the size and frequency of spherules may be associated with AMD.

Correction to: Optic nerve thinning and neurosensory retinal degeneration in the rTg4510 mouse model of frontotemporal dementia.

In the original publication of this article [1], the funding acknowledgement for grant "Alzheimer Society Research Program (ASRP) from the Alzheimer Society of Canada" was missing.

Optic nerve thinning and neurosensory retinal degeneration in the rTg4510 mouse model of frontotemporal dementia.

Visual impairments, such as difficulties in reading and finding objects, perceiving depth and structure from motion, and impaired stereopsis, have been reported in tauopathy disorders, such as frontotemporal dementia (FTD). These impairments however have been previously attributed to cortical pathologies rather than changes in the neurosensory retina or the optic nerve. Here, we examined tau pathology in the neurosensory retina of the rTg(tauP301L)4510 mouse model of FTD. Optic nerve pathology in mice was also assessed using MRI, and corresponding measurements taken in a cohort of five FTD sufferers and five healthy controls. rTg(tauP301L)4510 mice were imaged (T2-weighted MRI) prior to being terminally anesthetized and eyes and brains removed for immunohistochemical and histological analysis. Central and peripheral retinal labelling of tau and phosphorylated tau (pTau) was quantified and retinal layer thicknesses and cell numbers assessed. MR volumetric changes of specific brain regions and the optic nerve were compared to tau accumulation and cell loss in the visual pathway. In addition, the optic nerves of a cohort of healthy controls and behavioural variant FTD patients, were segmented from T1- and T2-weighted images for volumetric study. Accumulation of tau and pTau were observed in both the central and peripheral retinal ganglion cell (RGC), inner plexiform and inner nuclear layers of the neurosensory retina of rTg(tauP301L)4510 mice. This pathology was associated with reduced nuclear density (- 24.9 ± 3.4%) of the central RGC layer, and a reduced volume (- 19.3 ± 4.6%) and elevated T2 signal (+ 27.1 ± 1.8%) in the optic nerve of the transgenic mice. Significant atrophy of the cortex (containing the visual cortex) was observed but not in other area associated with visual processing, e.g. the lateral geniculate nucleus or superior colliculus. Atrophic changes in optic nerve volume were similarly observed in FTD patients (- 36.6 ± 2.6%). The association between tau-induced changes in the neurosensory retina and reduced optic nerve volume in mice, combined with the observation of optic nerve atrophy in clinical FTD suggests that ophthalmic tau pathology may also exist in the eyes of FTD patients. If tau pathology and neurodegeneration in the retina were to reflect the degree of cortical tau burden, then cost-effective and non-invasive imaging of the neurosensory retina could provide valuable biomarkers in tauopathy. Further work should aim to validate whether these observations are fully translatable to a clinical scenario, which would recommend follow-up retinal and optic nerve examination in FTD.

The clinical relevance of visualising the peripheral retina.

Recent developments in imaging technologies now allow the documentation, qualitative and quantitative evaluation of peripheral retinal lesions. As wide field retinal imaging, capturing both the central and peripheral retina up to 200° eccentricity, is becoming readily available the question is: what is it that we gain by imaging the periphery? Based on accumulating evidence it is clear that findings in the periphery do not always associate to those observed in the posterior pole. However, the newly acquired information may provide useful clues to previously unrecognised disease features and may facilitate more accurate disease prognostication. In this review, we explore the anatomy and physiology of the peripheral retina, focusing on how it differs from the posterior pole, recount the history of peripheral retinal imaging, describe various peripheral retinal lesions and evaluate the overall relevance of peripheral retinal findings to different diseases.

Peripheral Retinal Imaging Biomarkers for Alzheimer's Disease: A Pilot Study.

PURPOSE: To examine whether ultra-widefield (UWF) retinal imaging can identify biomarkers for Alzheimer's disease (AD) and its progression. METHODS: Images were taken using a UWF scanning laser ophthalmoscope (Optos P200C AF) to determine phenotypic variations in 59 patients with AD and 48 healthy controls at baseline (BL). All living participants were invited for a follow-up (FU) after 2 years and imaged again (if still able to participate). All participants had blood taken for genotyping at BL. Images were graded for the prevalence of age-related macular degeneration-like pathologies and retinal vascular parameters. Comparison between AD patients and controls was made using the Student t test and the χ2 test. RESULTS: Analysis at BL revealed a significantly higher prevalence of a hard drusen phenotype in the periphery of AD patients (14/55; 25.4%) compared to controls (2/48; 4.2%) [χ2 = 9.9, df = 4, p = 0.04]. A markedly increased drusen number was observed at the 2-year FU in patients with AD compared to controls. There was a significant increase in venular width gradient at BL (zone C: 8.425 × 10-3 ± 2.865 × 10-3 vs. 6.375 × 10-3 ± 1.532 × 10-3, p = 0.008; entire image: 8.235 × 10-3 ± 2.839 × 10-3 vs. 6.050 × 10-3 ± 1.414 × 10-3, p = 0.004) and a significant decrease in arterial fractal dimension in AD at BL (entire image: 1.250 ± 0.086 vs. 1.304 ± 0.089, p = 0.049) with a trend for both at FU. CONCLUSIONS: UWF retinal imaging revealed a significant association between AD and peripheral hard drusen formation and changes to the vasculature beyond the posterior pole, at BL and after clinical progression over 2 years, suggesting that monitoring pathological changes in the peripheral retina might become a valuable tool in AD monitoring.

Localization of the zinc binding tubulin polymerization promoting protein in the mice and human eye.

Tubulin Polymerization Promoting Protein (TPPP/p25) modulates the dynamics and stability of the microtubule network by its bundling and acetylation enhancing activities that can be modulated by the binding of zinc to TPPP/p25. Its expression is essential for the differentiation of oligodendrocytes, the major constituents of the myelin sheath, and has been associated with neuronal inclusions. In this paper, evidence is provided for the expression and localization of TPPP/p25 in the zinc-rich retina and in the oligodendrocytes in the optic nerve. Localization of TPPP/p25 was established by confocal microscopy using calbindin and synaptophysin as markers of specific striations in the inner plexiform layer (IPL) and presynaptic terminals, respectively. Postsynaptic nerve terminals in striations S1, S3 and S5 in the IPL and a subset of amacrine cells show immunopositivity against TPPP/p25 both in mice and human eyes. The co-localization of TPPP/p25 with acetylated tubulin was detected in amacrine cells, oligodendrocyte cell bodies and in synapses in the IPL. Quantitative Western blot revealed that the TPPP/p25 level in the retina was 0.05-0.13 ng/µg protein, comparable to that in the brain. There was a central (from optic nerve head) to peripheral retinal gradient in TPPP/p25 protein levels. Our in vivo studies revealed that the oral zinc supplementation of mice significantly increased TPPP/p25 as well as acetylated tubulin levels in the IPL. These results suggest that TPPP/p25, a microtubule stabilizer can play a role in the organization and reorganization of synaptic connections and visual integration in the eye.