Human Donor Eyes Research Articles

Biomechanical contribution of the sclera to dynamic corneal response in air-puff induced deformation in human donor eyes

This study was conducted to evaluate the impact of varying scleral material properties on the biomechanical response of the cornea under air-puff induced deformation. Twenty pairs of human donor eyes were obtained for this study. One eye from each pair had its sclera stiffened using 4% glutaraldehyde, while the fellow eye served as control for uniaxial strip testing. The whole globes were mounted in a rigid holder and intraocular pressure (IOP) was set using a saline column. Dynamic corneal response parameters were measured before and after scleral stiffening using the CorVis ST, a dynamic Scheimpflug analyzer. IOP was set to 10, 20, 30, and 40 mmHg, with at least 3 examinations performed at each pressure step. Uniaxial tensile testing data were fit to a neo-Hookean model to estimate the Young's modulus of treated and untreated sclera. Scleral Young's modulus was found to be significantly correlated with several response parameters, including Highest Concavity Deformation Amplitude, Peak Distance, Highest Concavity Radius, and Stiffness Parameter-Highest Concavity (SP-HC). There were significant increases in SP-HC after scleral stiffening at multiple levels of IOP, while no significant difference was observed in the corneal Stiffness Parameter – Applanation 1 (SP-A1) at any level of IOP. Scleral mechanical properties significantly influenced the corneal deformation response to an air-puff. The stiffer the sclera, the greater the constraining effect on corneal deformation resulting in lower displaced amplitude. This may have important clinical implications and suggests that both corneal and scleral material properties contribute to the observed corneal response in air-puff induced deformation.

Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth

Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1+ macrophages/microglia in human donor eye sections (aged and AMD) using multi-marker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD.

Single-cell transcriptomics of the human retinal pigment epithelium and choroid in health and macular degeneration

The human retinal pigment epithelium (RPE) and choroid are complex tissues that provide crucial support to the retina. Disease affecting either of these supportive tissues can lead to irreversible blindness in the setting of age-related macular degeneration. In this study, single-cell RNA sequencing was performed on macular and peripheral regions of RPE-choroid from 7 human donor eyes in 2 independent experiments. In the first experiment, total RPE/choroid preparations were evaluated and expression profiles specific to RPE and major choroidal cell populations were identified. As choroidal endothelial cells represent a minority of the total RPE/choroidal cell population but are strongly implicated in age-related macular degeneration (AMD) pathogenesis, a second single-cell RNA-sequencing experiment was performed using endothelial cells enriched by magnetic separation. In this second study, we identified gene expression signatures along the choroidal vascular tree, classifying the transcriptome of human choriocapillaris, arterial, and venous endothelial cells. We found that the choriocapillaris highly and specifically expresses the regulator of cell cycle gene (RGCC), a gene that responds to complement activation and induces apoptosis in endothelial cells. In addition, RGCC was the most up-regulated choriocapillaris gene in a donor diagnosed with AMD. These results provide a characterization of the human RPE and choriocapillaris transcriptome, offering potential insight into the mechanisms of choriocapillaris response to complement injury and choroidal vascular disease in age-related macular degeneration.

Investigation of factors associated with ABCB5-positive limbal stem cell isolation yields from human donors

PurposeTo identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. MethodsWhole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donor death. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors’ medical information. ResultsThe mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ± 160,000 and 2,608 ± 1,842 respectively, mean ± SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). ConclusionsOur study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.

Age, lipofuscin and melanin oxidation affect fundus near-infrared autofluorescence.

BackgroundFundus autofluorescence is a non-invasive imaging technique in ophthalmology. Conventionally, short-wavelength autofluorescence (SW-AF) is used for detection of lipofuscin, a byproduct of the visual cycle which accumulates with age or disease in the retinal pigment epithelium (RPE). Furthermore, near-infrared autofluorescence (NIR-AF) is used as a marker for RPE and choroidal melanin, but contribution of lipofuscin to the NIR-AF signal is unclear.MethodsWe employed fluorescence microscopy to investigate NIR-AF properties of melanosomes, lipofuscin and melanolipofuscin granules in histologic sections of wildtype and Abca4−/− mouse eyes, the latter having increased lipofuscin, as well as aged human donor eyes. Differentiation between these pigments was verified by analytical electron microscopy. To investigate the influence of oxidative and photic stress we used an in vitro model with isolated ocular melanosomes and an in vivo phototoxicity mouse model.FindingsWe show that NIR-AF is not an intrinsic property of melanin, but rather increases with age and after photic or oxidative stress in mice and isolated melanosomes. Furthermore, when lipofuscin levels are high, lipofuscin granules also show NIR-AF, as confirmed by correlative fluorescence and electron microscopy in human tissue. However, lipofuscin in albino Abca4−/− mice lacks NIR-AF signals.InterpretationWe suggest that NIR-AF is derived from melanin degradation products that accumulate with time in lipofuscin granules. These findings can help to improve the interpretation of patient fundus autofluorescence data.FundingThis work was supported by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft and Chinese Scholarship Council. Major instrumentation used in this work was supported by Deutsche Forschungsgemeinschaft, the European Fund for Regional Development and the state of Baden-Württemberg.

Age-related changes of the human retinal vessels: Possible involvement of lipid peroxidation

BackgroundAging of the human retina is accompanied by oxidative stress that exerts profound changes in the retinal neurons. It is unknown if oxidative stress influences the cellular components of the retinal vessels in some ways. MethodsWe examined changes in retinal vessels in human donor eyes (age: 35–94 years; N=18) by light and transmission electron microscopy, TUNEL and immunohistochemistry for biomarkers of vascular smooth muscle cells (SMC; actin), oxidative stress (4-hydroxy 2-nonenal [HNE] and nitrotyrosine), microglia (Iba-1) and vessels (isolectin B4). ResultsThe earliest changes in the endothelium and pericytes of capillaries are apparent from the seventh decade. With aging, there is clear loss of organelles and cytoplasmic filaments, and a progressive thickening of the endothelial and pericyte basal lamina. Loss of filaments, accumulation of lipofuscin and autophagic vacuoles are significant events in aging pericytes and SMC. Actin immunolabelling reveals discontinuity in arterial SMC layers during eighth decade, indicating partial degeneration of SMC. This is followed by hyalinization, with degeneration of the endothelium and SMC in arteries and arterioles of the nerve fibre layer (NFL) and ganglion cell layer in ninth decade. Iba-1 positive microglia were in close contact with the damaged vessels in inner retina, and their cytoplasm was rich in lysosomes. HNE immunoreactivity, but not of nitrotyrosine, was detected in aged vessels from seventh decade onwards, suggesting that lipid peroxidation is a major problem of aged vessels. However, TUNEL positivity seen during this period was limited to few arteries and venules of NFL. ConclusionThis study shows prominent age-related alterations of the pericytes and SMC of retinal vessels. These changes may limit the energy supply to the neurons and be responsible for age-related loss of neurons of the inner retina.

Elemental concentrations in Choroid-RPE and retina of human eyes with age-related macular degeneration

Heavy metals, metallic and toxic elements are reported to play an essential role in the complex multifactorial pathogenesis of age-related macular degeneration (AMD). This study was aimed to measure the concentrations of these elements in choroid-RPE and retina of human donor eyes with and without age-related macular degeneration associated changes. Human cadaver donor eyeballs were obtained from the CU Shah eye bank, Sankara Nethralaya Eye Hospital, India, after removal of the cornea. 39 control and 51 AMD donor eyes were used in this study. Alabama grading was done on the histopathological sections to identify early and late age-related macular degeneration changes. Concentrations of lead, cadmium, chromium, cobalt, nickel, arsenic and selenium were determined in choroid-RPE and retina using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Further, gene expression of oxidative stress-related genes, Nrf-2, HO-1, GCLC, GCLM, and detoxification related gene GSTpi was performed. The data were analyzed for statistical significance using Graph Pad® Prism 5 software. Donor eyes with early and late AMD had significantly higher levels of lead, cadmium, chromium, arsenic, and nickel in choroid-RPE and retina compared to the control eyes. Selenium was significantly increased in late AMD compared to control. No significant difference was observed in the levels of cobalt between eyes with and without AMD. Decreased transcript levels of oxidative stress-related genes were observed in the choroid-RPE and retinal tissues. Nrf-2 (p < 0.05), HO-1 and GCLC expressions were lowered in the retina of AMD, whereas GCLM and GSTpi expressions were decreased (p < 0.05) with an increase in HO-1 in choroid-RPE of AMD. This study provides evidence that alterations of the heavy metals and toxic elements along with oxidative stress may play a role in the pathogenesis of AMD.

The Role of Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole Formation: A 3D Electron Microscopy Study.

PurposeWe investigated whether cellular connectivity between Schlemm's canal (SC) inner wall (IW) endothelium, and juxtacanalicular connective tissue (JCT), and between IW endothelial cells, plays a role in giant vacuole (GV) and pore formation by comparing perfusion- and immersion-fixed eyes.MethodsNormal human donor eyes (n = 4) were either immersion-fixed (0 mm Hg) or perfusion-fixed (15 mm Hg). Trabecular meshwork near SC was imaged using serial block-face scanning electron microscopy. A total of 12 IW cells from each group were 3D-reconstructed from ∼7040 electron micrographs and compared. In each cell, connections between IW cells and JCT cells/matrix were quantified; IW/IW connectivity was measured by cell border overlap length. GV volume, density, shape, and intracellular and paracellular pores were analyzed.ResultsThe mean number of IW/JCT cell-cell connections per cell significantly decreased (P < 0.01) while the summed GV volume per cell significantly increased (P < 0.01) in perfusion-fixed eyes compared to immersion-fixed eyes. Intracellular pores were observed in 14.6% of GVs in perfusion-fixed eyes and not observed in immersion-fixed eyes. The mean IW/IW overlap length per cell decreased (P < 0.01), and paracellular pores were found only in regions where IW/IW connectivity was minimal (overlap length = 0 μm) in perfusion-fixed eyes and not observed in immersion-fixed eyes.ConclusionsOur data suggest that changes in IW/JCT connectivity may be an important factor in the formation of larger GVs, and decreased IW/IW connectivity may promote paracellular pore formation. Targeting the IW/JCT and IW/IW connectivity may therefore be a potential strategy to regulate outflow resistance and IOP.

Donor-Dependent Factors Influencing Contamination Rates of Conjunctival Swabs of Human Donor Eyes.

BackgroundThis study investigated the effect of donor-dependent factors on contamination rates of conjunctival swabs of human donor eyes.Material/MethodsFrom July 2015 to September 2017 a total of 1008 conjunctival swabs from 504 consecutive human donor eyes were analyzed. Cross-tabulation, chi-squared tests, and Fisher’s exact tests were used to evaluate the effect of donor-dependent factors on contamination rates of conjunctival swabs.ResultsThe mean conjunctival swabs contamination rate was 28.4%. Donors with the diagnosis of carcinoma or metastases were associated with an increased conjunctival swab contamination rate [odds ratio (OR)=1.8, 95% confidence interval (CI)=1.2–2.6, p=0.007; OR=1.7, 95% CI=1.1–2.6, p=0.016; respectively].However, the age, sex, diagnosis of diabetes mellitus, and donors who received chemotherapy did not significantly increase the conjunctival swab contamination risk.ConclusionsDonors with the diagnosis of a carcinoma or metastases seemed to be predisposed to increased conjunctival swab contamination risk.

The Utah Protocol for Postmortem Eye Phenotyping and Molecular Biochemical Analysis.

PurposeCurrent understanding of local disease pathophysiology in AMD is limited. Analysis of the human disease-affected tissue is most informative, as gene expression, expressed quantitative trait loci, microenvironmental, and epigenetic changes can be tissue, cell type, and location specific. Development of a novel translational treatment and prevention strategies particularly for earlier forms of AMD are needed, although access to human ocular tissue analysis is challenging. We present a standardized protocol to study rapidly processed postmortem donor eyes for molecular biochemical and genomic studies.MethodsWe partnered with the Utah Lions Eye Bank to obtain donor human eyes, blood, and vitreous, within 6 hours postmortem. Phenotypic analysis was performed using spectral-domain optical coherence tomography (SD-OCT) and color fundus photography. Macular and extramacular tissues were immediately isolated, and the neural retina and retinal pigment epithelium/choroid from each specimen were separated and preserved. Ocular disease phenotype was analyzed using clinically relevant grading criteria by a group of four ophthalmologists incorporating data from SD-OCT retinal images, fundus photographs, and medical records.ResultsThe use of multimodal imaging leads to greater resolution of retinal pathology, allowing greater phenotypic rigor for both interobserver phenotype and known clinical diagnoses. Further, our analysis resulted in excellent quality RNA, which demonstrated appropriate tissue segregation.ConclusionsThe Utah protocol is a standardized methodology for analysis of disease mechanisms in AMD. It uniquely allows for simultaneous rigorous phenotypic, molecular biochemical, and genomic analysis of both systemic and local tissues. This better enables the development of disease biomarkers and therapeutic interventions.

Wnt Signaling Is Required for the Maintenance of Human Limbal Stem/Progenitor Cells In Vitro.

PurposeA chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).MethodsLSCs were isolated from human donor eyes and cultured as single cells for 12 to 14 days with the following small molecules: IIIC3, an antagonist of the Wnt signaling inhibitor Dickkopf (DKK), and IC15, a Wnt signaling inhibitor. Proliferation of LSCs in the presence of IIIC3 and IC15 was determined by the number of cells and colonies established. Maintenance of stemness was determined by p63α, cytokeratin (K)12, and K14 expression.ResultsActivation of Wnt, through IIIC3-mediated DKK inhibition, resulted in similar colony forming efficiency (CFE) as in the untreated LSCs, but significantly increased the number of cultivated cells 7.21% with 5 μM. Inhibition of Wnt with IC15 significantly reduced the CFE (P ≤ 0.01) and the number of cultivated cells by 16% to 29%. Percentage of cells expressing high levels of p63α (p63αbright) and quantity of small cells (≤12 μm), which contain the LSCs, increased 4.71% and 11.26% (both P < 0.05), respectively, with 5 μM IIIC3. All concentrations of IIIC3 and IC15 retained the K14 undifferentiated marker (97%), while differentiation, as detected by expression of K12, was found in up to 2% of cells in 1 μM IIIC3, 1 μM IC15, or 5 μM IIIC3.ConclusionsWnt signaling is required in LSC proliferation and maintenance of an undifferentiated state. The current study is a proof of concept that the Wnt pathway could be modulated in LSCs to enhance or decrease the efficiency of human LSC expansion.

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes

BackgroundAge-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation.MethodsEpigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes.ResultsGenome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10−9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10−7), and Tenascin X (TNXB) (p = 6.30 × 10−6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors.ConclusionsWe report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

Biomechanical Impact of the Sclera on Corneal Deformation Response to an Air-Puff: A Finite-Element Study.

Aim or Purpose: To describe the effect of varying scleral stiffness on the biomechanical deformation response of the cornea under air-puff loading via a finite-element (FE) model.Methods: A two-dimensional axisymmetric stationary FE model of the whole human eye was used to examine the effects varying scleral stiffness and intraocular pressure (IOP) on the maximum apical displacement of the cornea. The model was comprised of the cornea, sclera, vitreous, and surrounding air region. The velocity and pressure profiles of an air-puff from a dynamic Scheimpflug analyzer were replicated in the FE model, and the resultant profile was applied to deform the cornea in a multiphysics study (where the air-puff was first simulated before being applied to the corneal surface). IOP was simulated as a uniform pressure on the globe interior. The simulation results were compared to data from ex vivo scleral stiffening experiments with human donor globes.Results: The FE model predicted decreased maximum apical displacement with increased IOP and increased ratio of scleral-to-corneal Young's moduli. These predictions were in good agreement (within one standard deviation) with findings from ex vivo scleral stiffening experiments using human donor eyes. These findings demonstrate the importance of scleral material properties on the biomechanical deformation response of the cornea in air-puff induced deformation.Conclusion: The results of an air-puff induced deformation are often considered to be solely due to IOP and corneal properties. The current study showed that the stiffer the sclera, the greater will be the limitation on corneal deformation, separately from IOP. This may have important clinical implications to interpreting the response of the cornea under air-puff loading in pathologic conditions.

An In Vitro Human Lens Capsular Bag Model Adopting a Graded Culture Regime to Assess Putative Impact of IOLs on PCO Formation.

To develop a culture regime for the in vitro human lens capsular bag model that better reflects clinical events following cataract surgery and to use this refined model to evaluate the putative impact of IOLs on PCO formation. Capsulorhexis and lens extraction were performed on human donor eyes to generate capsular bags attached to the ciliary body by the zonules. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining in 6 mL serum-free EMEM for 28 days or in a graded culture system (days 1-3, 5% human serum and 10 ng/mL TGFβ2; days 4-7, 2% human serum and 1 ng/mL TGFβ2; days 8-14, 1% human serum and 0.1 ng/mL TGFβ2; days 15-28, serum-free EMEM), which better mimics clinical changes. Preparations were monitored with phase-contrast and modified-dark-field microscopy. Cell coverage and light scatter were quantified using image analysis software. The transdifferentiation marker, α-SMA and matrix component, fibronectin were assessed by immunocytochemistry. To assess IOLs in the model, Alcon Acrysof or Hoya Vivinex IOLs were implanted in match-paired capsular bags. Match-paired experiments showed that graded culture enhanced growth, facilitated matrix contraction, increased transdifferentiation, and promoted matrix deposition relative to serum-free culture. The graded culture protocol was applied to match-paired bags implanted with a Hoya Vivinex or an Alcon Acrysof IOL. The Vivinex demonstrated a lag in growth across the posterior capsule. However, by day 28, coverage was similar, but light-scatter was greater with Acrysof implanted. Cell growth on the Acrysof IOL anterior surface was significantly greater than Vivinex. The graded culture human capsular bag model serves as an excellent system to evaluate and develop intraocular lenses. The Hoya Vivinex IOL showed an overall better level of performance against postsurgical wound healing and PCO than the Alcon Acrysof using this model.

Metformin activity in an in vitro model of posterior capsule opacification

Purpose: To determine the activity of metformin in an in vitro model of posterior capsule opacification (PCO).Design: Experimental laboratory research. Methods: The HLE-B3 lens epithelial cell line was treated with PCO induction media (PCOM) supplemented with transforming growth factor-beta (TGF-β) and fibroblast growth factor (FGF). Different metformin concentrations (0-100 mM) were used. The following cellular parameters were assessed: (1) survival, using a viability assay; (2) morphology, via microscopy and image analysis; (3) migration, using the wound assay; (4) and expression of epithelial (Pax6, E-cadherin) and mesenchymal (α-smooth muscle actin or α-SMA, fibronectin) markers via Western blot. Expression of the uptake receptor SLC22A1 was evaluated in HLE-B3 and in human donor eyes with Western blot and immunohistochemistry, respectively. Statistical analysis of variance (ANOVA) with Tukey post-hoc test was done for analysis of cytotoxicity, morphology and migration data. Results: Metformin was lethal to half (LC50) of the cells at 30 mM, and a decrease in viability (P&lt;0.05) was noted at 5 mM. LECs in PCOM treated with 1 mM metformin showed increased Pax6 and E-cadherin and decreased α-SMA and fibronectin expression. LECs in PCOM treated with metformin also maintained epithelial morphology. Migration was inhibited with 0.5 mM metformin (P&lt;0.05). Both HLE-B3 and the lens epithelium in donor eyes were found to express SLC22A1.Conclusion: Metformin decreased survival and migration in LECs, maintaining epithelial phenotype and reducing mesenchymal marker expression. Metformin therefore has potential as an adjunct in PCO prevention.Financial Disclosures: This work was partially funded by Mitacs Canada.

Characterization of a tissue-engineered choroid

The choroid of the eye is a vascularized and pigmented connective tissue lying between the retina and the sclera. Increasing evidence demonstrates that, beyond supplying nutrients to the outer retina, the different choroidal cells contribute to the retina’s homeostasis, especially by paracrine signaling. However, the precise role of each cell type is currently unclear. Here, we developed a choroidal substitute using the self-assembly approach of tissue engineering. Retinal pigment epithelial (RPE) cells, as well as choroidal stromal fibroblasts, vascular endothelial cells and melanocytes, were isolated from human eye bank donor eyes. Fibroblasts were cultured in a medium containing serum and ascorbic acid. After six weeks, cells formed sheets of extracellular matrix (ECM), which were stacked to produce a tissue-engineered choroidal stroma (TECS). These stromal substitutes were then characterized and compared to the native choroid. Their ECM composition (collagens and proteoglycans) and biomechanical properties (ultimate tensile strength, strain and elasticity) were similar. Furthermore, RPE cells, human umbilical vein endothelial cells and choroidal melanocytes successfully repopulated the stromas. Physiological structures were established, such as a confluent monolayer of RPE cells, vascular-like structures and a pigmentation of the stroma. Our TECS thus recaptured the biophysical environment of the native choroid, and can serve as study models to understand the normal interactions between the RPE and choroidal cells, as well as their reciprocal exchanges with the ECM. This will consequently pave the way to derive accurate insight in the pathophysiological mechanisms of diseases affecting the choroid. Statement of significanceThe choroid is traditionally known for supplying blood to the avascular outer retina. There has been a renewed attention directed towards the choroid partly due to its implication in the development of age-related macular degeneration (AMD), the leading cause of blindness in industrialized countries. Since AMD involves the dysfunction of the choroid/retinal pigment epithelium (RPE) complex, a three-dimensional (3D) model of RPE comprising the choroid layer is warranted. We used human choroidal cells to engineer a choroidal substitute. Our approach takes advantage of the ability of cells to recreate their own environment, without exogenous materials. Our model could help to better understand the role of each choroidal cell type as well as to advance the development of new therapeutics for AMD.

Histological investigation of human glaucomatous eyes: Extracellular fibrotic changes and galectin 3 expression in the trabecular meshwork and optic nerve head.

Glaucoma is a leading cause of irreversible vision loss and is associated with fibrotic changes in two ocular tissues-the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences in extracellular matrix components (ECM) including collagen, elastin, transforming growth factor beta-2, type-II receptor (TGFβRII) and Galectin3 (Gal3) in the glaucomatous human eyes to quantify fibrotic changes in ONH and TM. Glaucomatous and control human donor eyes were prepared for chemical and immunological staining to quantify ECM protein expression in the TM and ONH. Chemical staining included: Trichrome (collagen), Vernhoeff-Van Giesen (elastin) and Sirius Red (collagen). Immunohistochemistry was used to determine levels of Gal3 and TGFβ2RII. Quantitative analyses were performed using Image J software. Student's t-test was used to compare groups and Pearson's test was used to determine correlations P-values of 0.05 (or less) were considered statistically significant. Deposition of ECM proteins was elevated in glaucomatous tissues. There was increased collagen (P = 0.0469), Gal3 (P < 0.0001) and TGFβ2RII (P = 0.0005) in the TM of glaucomatous eyes. Likewise, collagen (P = 0.0517) and Galectin3 (P = 0.041) were increased in the ONH glaucomatous eyes. There was a correlation of TGFβRII with Gal3 in the TM (P < 0.0001) and optic nerve (P = 0.0003). The TM and ONH of glaucomatous eyes showed increased expression of ECM proteins supporting a fibrotic pathology. Galectin3 and TGFβ-2R II showed a positive correlation in TM and optic nerve supporting co-localization and suggesting their potential role in the glaucoma fibrotic process. Clin. Anat. 31:1031-1049, 2018. © 2018 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Factors influencing the contamination rates of the conjunctival swabs and organ culture media of human donor eyes: Erratum.

Factors influencing the contamination rates of the conjunctival swabs and organ culture media of human donor eyes: Erratum.

Three-Dimensional Microscopy Demonstrates Series and Parallel Organization of Human Peripapillary Capillary Plexuses.

To define the three-dimensional topologic organization of the human peripapillary capillary plexuses in order to better understand the hemodynamic characteristics of this retinal circulation. The retinal microvasculature was perfusion labeled in five normal human donor eyes, and optical stacks were collected from regions immediately superior, temporal, inferior, and nasal to the optic disk by using confocal scanning laser microscopy. The spatial location and morphometric characteristics of capillary plexuses were compared. Three-dimensional visualization strategies were used to document the organization of vascular conduits that interconnect capillary beds and to study the communications between capillary beds and precapillary arterioles and postcapillary venules. The peripapillary microcirculation is composed of four capillary plexuses, including the radial peripapillary capillary plexus at the level of the nerve fiber layer, the superficial capillary plexus (SCP) at the level of the ganglion cell layer, the intermediate capillary plexus located at the inner boundary of the inner nuclear layer, and the deep capillary plexus located at the outer boundary of the inner nuclear layer. Capillary diameter and density were significantly different between the four plexuses (both P ≤ 0.011). The SCP is the only capillary bed that receives feeding and draining branches directly from precapillary arterioles and postcapillary venules, respectively. Four different inflow and outflow patterns characterized the communication between the SCP and surrounding capillary beds. The capillary plexuses of the human peripapillary microcirculation are arranged in series and parallel and manifest specializations that likely reflect the unique metabolic demands and biochemical environment of the retinal layers.

Factors influencing the contamination rates of the conjunctival swabs and organ culture media of human donor eyes

This study assessed the influence of donor, environmental, and logistical factors on the contamination rates of the conjunctival swabs and organ culture media of human donor eyes.In total, 1008 conjunctival swabs and 418 organ culture media samples from 504 consecutive human donor eyes were analyzed. Cross-tabulation, chi-squared tests, and Fisher's exact tests were used to assess the influence of the different factors on the contamination rates of the conjunctival swabs and organ culture media.The overall contamination rates were 28.4% for the conjunctival swabs and 1.0% for the organ culture media. A prolonged time between death and the conjunctival swab collection was associated with an increased conjunctival swab contamination rate [odds ratio (OR) = 1.9, 95% confidence interval (CI) = 1.2–3.0, P = .007]. The highest conjunctival swab contamination rate was found in the corneas procured in external institutions (outside the university hospital) (44.1%, OR = 3.6, 95%CI = 1.5–8.4, P = .003). Hospitalization times of 2 to 7 days prior to death were associated with an increased conjunctival swab contamination risk (OR = 2.6, 95%CI = 1.1–5.8, P = .021). However, the sex, age, cause of donor death, differentiation between septic and aseptic donors, differentiation between heart-beating brain-dead multiorgan donors and cadaveric donors, a warmer mean monthly temperature, and death to corneoscleral disc excision time did not significantly increase the conjunctival swab contamination risk. In addition, none of these factors affected the organ culture media contamination risk. Moreover, a positive conjunctival swab did not significantly increase the media contamination risk (P = .08). Surprisingly, the microorganisms causing media contamination were present at 50% of the amount detected on the conjunctival surface of the respective donor eye.A prolonged time between death and the conjunctival swab collection, a hospitalization time of 2 to 7 days prior to death, and corneal collection outside the university hospital seemed to be the main factors responsible for an increased conjunctival swab contamination risk. In addition, our investigation illustrated that a positive conjunctival swab is not a strong indicator for organ culture media contamination. Critical discussion is necessary regarding the validity of conjunctival swabs as prognostic parameters for organ culture media contamination.