Donor Eyes Research Articles

Spatial targeted delivery of riboflavin with a controlled corneal iontophoresis delivery system in theranostic-guided UV-A light photo-therapy.

Seven human donor eye globes underwent corneal cross-linking using theranostic UV-A device with accessory corneal iontophoresis system for patterned delivery of a 0.22% riboflavin solution. Theranostic-guided UV-A light illumination assessed riboflavin distribution and treated corneas at 10 mW/cm2 for 9 min with a 5.0-mm beam size. Corneal topography maps were taken at baseline and 2-h post-treatment. Analysis utilized corneal topography elevation data, with results showing controlled riboflavin delivery led to a consistent gradient, with 40% higher levels centrally (248 ± 79 μg/cm3) than peripherally (180 ± 72 μg/cm3 at ±2.5 mm from the center). Theranostic-guided UV-A light irradiation resulted in significant changes in corneal topography, with a decrease in best-fit sphere value (-0.7 ± 0.2 D; p < 0.001) and consistent downward shift in corneal elevation map (-11.7 ± 3.7 μm). The coefficient of variation was 2.5%, indicating high procedure performance in achieving significant and reliable corneal flattening.

Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus.

The purpose of this study was to determine if levels of the HtrA1 protein in serum or vitreous humor are influenced by genetic risk for age-related macular degeneration (AMD) at the 10q26 locus, age, sex, AMD status, and/or AMD disease severity, and, therefore, to determine the contribution of systemic and ocular HtrA1 to the AMD disease process. A custom-made sandwich ELISA assay (SCTM ELISA) for detection of the HtrA1 protein was designed and compared with three commercial assays (R&D Systems, MyBiosource 1 and MyBiosource 2) using 65 serum samples. Concentrations of HtrA1 were thereafter determined in serum and vitreous samples collected from 248 individuals and 145 human donor eyes, respectively. The SCTM ELISA demonstrated high specificity, good recovery, and parallelism within its linear detection range and performed comparably to the R&D Systems assay. In contrast, we were unable to demonstrate the specificity of the two assays from MyBioSource using either recombinant or native HtrA1. Analyses of concentrations obtained using the validated SCTM assay revealed that genetic risk at the 10q26 locus, age, sex, or AMD status are not significantly associated with altered levels of the HtrA1 protein in serum or in vitreous humor (P > 0.05). HtrA1 levels in serum and vitreous do not reflect the risk for AMD associated with the 10q26 locus or disease status. Localized alteration in HTRA1 expression in the retinal pigment epithelium, rather than systemic changes in HtrA1, is the most likely driver of elevated risk for developing AMD among individuals with risk variants at the 10q26 locus.

ANGPTL7 and Its Role in IOP and Glaucoma.

Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics. IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured. IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies. Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.

Granzyme B degrades extracellular matrix and promotes inflammation and choroidal neovascularization

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.

Quality and applicability of cadaveric donor eyes for molecular biology research: An Indian experience.

Human ocular tissue banking plays an important part in the advancement of translational research for identifying the molecular processes involved in disease etiology and pathogenesis. Timely obtaining a good-quality ocular tissue from a cadaveric donor is exceedingly difficult, especially in remote areas, with a variable transportation time (within 12-24 h), raising concerns about RNA quality and its subsequent applications. Therefore, we assessed the utility of retinal tissues from cadaver donor and enucleated eyes based on the RNA quality and gene expression by real-time polymerase chain reaction (PCR). Prospective study. Retina tissues were separated from the donor/enucleated eyes received in the eye bank within 24 h of death (n = 15) and within an hour from OR (n = 3), respectively, and stored immediately at -80 degree. RNA was isolated using trizol, and the quantity and quality were assessed using Qubit and agarose gel electrophoresis, respectively. QPCR was performed for measuring the expression of different retinal-specific genes. The cellular viability of the retina was assessed by establishing explant primary cell cultures. The data were calculated as an average of normalised Ct values ± standard error of the mean. RNA obtained from cadaveric tissues despite being partially degraded showed a uniform strong gene expression of several retinal-specific genes such as PAX6, RHO, TUBB3, CRX , and ALDH1L1 . The primary cultures established from cadaveric tissues showed viable cells. The cadaver donor tissues collected within 24 hours of death can be effectively utilized for gene expression profiling.

Influence of Donor Factors on Descemet Membrane Endothelial Keratoplasty (DMEK) Graft Preparation Outcome.

To determine which donor characteristics, like previous diseases and surgeries, influence the severity of the DM/endothelial lamella preparation prior to DMEK-surgery. Retrospective cross-sectional single-center study is presented. Eight hundred and forty-six eyes with DMEK-surgery between 01/2018 and 01/2021 performed at the University Hospital Cologne, Germany, were included. Information regarding the donors' previous diseases and surgeries were provided by a large database of a cornea bank (Multi Tissue Bank Mecklenburg-Vorpommern) and merged with the Cologne DMEK database, which contains information regarding preparation characteristics of the surgeon-prepared graft directly preoperatively. Three preparation groups (easy, difficult and very difficult) were correlated to the donors' previous diseases and surgeries. The following characteristics were used for the assignment in one of the three groups: stripping difficulty, rolling and staining behavior, central and peripheral adherences, tissue fragility and DM-splitting. Significant risk factors for DM-splitting were diabetes mellitus (DMel) type II, heart failure, chronic kidney disease and previous cataract surgery (p=0.022, p=0.012; p=0.047 and p<0.001 respectively). Previous DMel (especially type 2) was significantly associated with the occurrence of central adherences (p=0.009). Several cardiovascular diseases (p-values between <0.001 and p=0.038), DMel type II, chronic kidney disease and previous cataract-surgery were associated with peripheral adherences (p=0.004; p=0.020 and p<0.001 respectively). Furthermore, pseudophakic donor eyes presented a higher degree of fragility of the graft (p<0.001). Age was a significant risk factor for difficult preparation (p<0.001). The staining of the graft was poorer in donors with chronic kidney disease (p=0.037). Donor diabetes mellitus type 2, heart failure, previous cataract surgery, chronic kidney disease and age are associated with a difficult DMEK graft preparation. For every one-year increment in donor age, the odds of having very difficult preparation were increased by 3%. Also, chronic kidney disease predisposes to a poor tissue staining with trypan blue during preparation.

Long-term ocular surface stability in conjunctivolimbal autograft and ocular surface safety in the donor eyes.

The current study is aimed to present the long-term results of the patients who underwent conjunctivolimbal autograft (CLAU) as the primary operation in unilateral limbal stem cell deficiency and the ocular surface safety of the donor eyes. The patients were followed up for fiveyears or longer. The records of all patients who underwent CLAU as the primary operation were retrospectively analyzed. Additional ocular surface operations, ocular surface stability, best-corrected visual acuity (BCVA), and ocular surface status of the donor eyes were investigated. The mean age of the patients at the time of transplantation was 35.07 ± 12.9 (12-60). Twenty-nine eyes of 29 patients were followed up for an average of 97.82 ± 34.45 (60-186) months. Additional ocular surface operation was required in 27.58% (8/29) of the eyes in order to achieve a stable ocular surface. Ocular surface stability was achieved in 82.75% (24/29) of the eyes at the end of the follow-up period. BCVA increased from 1.78 ± 0.82 to 0.91 ± 0.92 logMAR at the last visit (p < 0.001). Corneal ectasia and vascularization developed in one donor eye in the fifth postoperative year. CLAU tissues provide ocular surface stability with a successful vision result in the long term. CLAU theoretically carries risks including limbal stem cell deficiency in the donor eye. In the long-term follow-up of donor eyes after CLAU, ectasia and limbal stem cell deficiency were observed in one eye.

Impact of demographic factors on corneal donor recovery.

Death-to-preservation time (DTP) is a commonly reported, but infrequently studied, measure of efficiency for the corneal tissue procurement process and is a key screening component for corneal tissue suitability for transplantation. It is unknown whether demographic factors such as race, age, or gender may affect DTP. This retrospective cross-sectional study included all deceased-donor eye tissue collected by CorneaGen Eye Banks between June 1, 2012 and June 30, 2016. Exposure variables of race, age, and gender were independently analyzed with the outcome variable, DTP, using three simple linear regression analyzes.Associations were then confirmed by a multiple linear regression analysis within a single model. A total of 24,138 unique donors were identified from 48,207 donor eyes. Simple linear regression analysis showed that relative to White donors, Black and Hispanic donors were associated with a 2.40h (95% CI 2.07-2.74h, p < 0.001) and 2.48h (95% CI 2.15-2.80h, p < 0.001) longer mean DTP, respectively. DTP decreased with increasing age, at a rate of 30min per every 10years (95% CI 27-33min, p < 0.001).Male donors were associated with a 35min (95% CI 26-44min, p < 0.001) longer DTP relative to female donors. A multiple linear regression confirmed the results of the three simple linear regressions. In a large cohort of corneal donors, non-White race, younger age, and male gender were associated with longer DTP.

Calpain-specific breakdown fragment in human drusen.

With aging and age-related macular dystrophy (AMD), proteolytic fragments are deposited in extracellular drusen located between the RPE and Bruch's membrane. Localized hypoxia may be a risk factor for AMD. Our hypothesis is that following hypoxia, activation of proteolytic enzymes called calpains may cause proteolysis/degeneration of retinal cells and RPE. No direct evidence has yet demonstrated activation of calpains in AMD. The purpose of the present study was to identify calpain-cleaved proteins in drusen. Seventy-six (76) drusen were analyzed in human eye sections from six normal and twelve AMD human donor eyes. The sections were subjected to immunofluorescence for the calpain-specific 150 kDa breakdown product from α-spectrin, SBDP150 - a marker for calpain activation, and for recoverin - a marker for photoreceptor cells. Among 29 nodular drusen, 80% from normal eyes and 90% from AMD eyes stained positive for SBDP150. Among 47 soft drusen, mostly from AMD eyes, 72% stained positive for SBDP150. Thus, the majority of both soft and nodular drusen from AMD donors contained SBDP150. SBDP150 was detected for the first time in soft and nodular drusen from human donors. Our results suggest that calpain-induced proteolysis participates in the degeneration of photoreceptors and/or RPE cells during aging and AMD. Calpain inhibitors may ameliorate AMD progression.

Genome-wide association identifies novel ROP risk loci in a multiethnic cohort

We conducted a genome-wide association study (GWAS) in a multiethnic cohort of 920 at-risk infants for retinopathy of prematurity (ROP), a major cause of childhood blindness, identifying 1 locus at genome-wide significance level (p < 5×10-8) and 9 with significance of p < 5×10-6 for ROP ≥ stage 3. The most significant locus, rs2058019, reached genome-wide significance within the full multiethnic cohort (p = 4.96×10-9); Hispanic and European Ancestry infants driving the association. The lead single nucleotide polymorphism (SNP) falls in an intronic region within the Glioma-associated oncogene family zinc finger 3 (GLI3) gene. Relevance for GLI3 and other top-associated genes to human ocular disease was substantiated through in-silico extension analyses, genetic risk score analysis and expression profiling in human donor eye tissues. Thus, we identify a novel locus at GLI3 with relevance to retinal biology, supporting genetic susceptibilities for ROP risk with possible variability by race and ethnicity.

Viability of Transplanted De Novo Retinal Ganglion Cells in Human Donor Eyes Maintained Under Elevated Intraocular Pressure

Purpose: Glaucoma is a group of optic neuropathies characterized by visual field loss, classically due to increased intraocular pressure (IOP) and retinal ganglion cell (RGC) degeneration. Current treatment options reduce IOP, but RGC degeneration persists. De novoRGCs can be differentiated from reprogrammed human corneal fibroblasts and transplanted into the retina to potentially restore vision in patients with late-stage disease when most RGCs are irreversibly damaged. We investigate the survival of these human induced pluripotent stem cell (IPSC) derived RGCs after culturing them in human donor eyes under conditions of elevated and normal IOP using the ocular translaminar autonomous system (TAS) chamber.&#x0D; Methods: Human iPSCs were generated by reprogramming human donor corneal fibroblasts using Sendai viral vectors with Yamanaka factors. These iPSCs were then differentiated into retinal organoids from which RGCs were obtained. The RGCs were transduced with AAV2-GFP and transplanted into donor human eyes obtained from control individuals. They were pressurized for approximately 5 days, with the left eye maintained at normal IOP and right eye at elevated IOP. Viability was measured by expression levels of pro-survival pathways via qRTPCR, immunohistochemistry staining, and electroretinography for retinal function (ERG).&#x0D; Results: We successfully transplanted human RGCs into human donor eyes and visualized them after GFP transduction. We maintained a pressure differential between the two eyes for approximately 5 days using the TAS model. Differential expression of survival, inflammatory and apoptotic genes was identified between normal and high IOP. We identified retinal function changes under normal and high IOP after RGC transplantation.&#x0D; Conclusions: Human iPSC derived RGCs provide a potential strategy to overcome vision loss in patients with diseases that damage RGCs such as glaucoma, Parkinson’s, Alzheimer's, multiple sclerosis, and traumatic optic neuropathy. Future investigation would involve integration of transplanted RGCs and directing their axons towards visual centers in the brain.

Spectral Analysis of Human Retinal Pigment Epithelium Cells in Healthy and AMD Eyes.

Retinal pigment epithelium (RPE) cells show strong autofluorescence (AF). Here, we characterize the AF spectra of individual RPE cells in healthy eyes and those affected by age-related macular degeneration (AMD) and investigate associations between AF spectral response and the number of intracellular AF granules per cell. RPE-Bruch's membrane flatmounts of 22 human donor eyes, including seven AMD-affected eyes (early AMD, three; geographic atrophy, one; neovascular, three) and 15 unaffected macula (<51years, eight; >80years, seven), were imaged at the fovea, perifovea, and near-periphery using confocal AF microscopy (excitation 488nm), and emission spectra were recorded (500-710nm). RPE cells were manually segmented with computer assistance and stratified by disease status, and emission spectra were analyzed using cubic spline transforms. Intracellular granules were manually counted and classified. Linear mixed models were used to investigate associations between spectra and the number of intracellular granules. Spectra of 5549 RPE cells were recorded. The spectra of RPE cells in healthy eyes showed similar emission curves that peaked at 580nm for fovea and perifovea and at 575 and 580nm for near-periphery. RPE spectral curves in AMD eyes differed significantly, being blue shifted by 10nm toward shorter wavelengths. No significant association coefficients were found between wavelengths and granule counts. This large series of RPE cell emission spectra at precisely predefined retinal locations showed a hypsochromic spectral shift in AMD. Combining different microscopy techniques, our work has identified cellular RPE spectral AF and subcellular granule properties that will inform future in vivo investigations using single-cell imaging.

Cytomegalovirus detected by qPCR in iris and ciliary body of immunocompetent corneal donors

BackgroundCytomegalovirus (CMV) can cause a wide panel of ocular infections. The involvement of CMV as a cause of anterior uveitis in the immunocompetent patient is recent and remains poorly understood. ObjectiveTo investigate the presence of CMV in anterior uveal tissues of immunocompetent corneal donors. Study DesignWe collected aqueous humor, iris, and ciliary body from both eyes of 25 donors died at the Limoges University Hospital between January 2020 and July 2021. CMV serology was determined for all patients from post-mortem blood sample. Ocular tissues were split in 2 fragments for qPCR and 2 for histological analysis. CMV genomes copies were quantified by Multiplex qPCR after DNA extraction. Results16 of 25 patients (64%) displayed positive CMV serology, with a median age of 67 years. Viremia was positive in 3 of 16 (19%) CMV-positive patients. No CMV DNA copies were found from the aqueous humor samples. CMV DNA was detected in iris and ciliary body of 28 of 32 eyes of seropositive donors, and 5 of 18 eyes of seronegative donors. The median viral copy number [IQR] was 2.41 × 102 [8.91 × 101 - 1.01 × 103] copies/1 × 106 cells in the CMV-positive group and 0.00 [0.00 - 3.54 × 102] copies/1 × 106 cells in the CMV-negative group (p<0.001). Histology and immunohistochemistry did not reveal any CMV lesions from any sample. ConclusionCMV DNA was found in iris and ciliary body of immunocompetent seropositive patients, but also, although less frequently, from seronegative donors. These results highlight mechanisms of infection, latency and reactivation of CMV in ocular tissues.

Insights into human choroid melanocyte and stromal cells

Aims/Purpose: The human choroid is a complex highly vascular pigmented tissue between Bruch's membrane and the sclera. Its dynamic nature is important for eye homeostasis and for in pathology including macular degeneration and myopia. The stroma comprises heterogeneous cell populations including fibroblasts, melanocytes, immune cells, smooth muscle cells and neurons within a loose collagenous extracellular matrix. We used RNA transcriptome and multimarker immunolabelling of human choroidal melanocytes and tissue to further characterize adult choroidal melanocytes.Methods: Human choroidal melanocytes were cultured (n = 6 postmortem donor eyes, &lt;18 h delay, UNSW HREC), and used between passage (P) 2 to 5 for RNA transcriptome and multimarker immunofluorescence labelling and confocal microscopy. Sections and flatmounts from central choroid were also compared. Immunolabelling included melanocyte (Tyrosinase, TRYP1, DCT, MelanA) and stromal cell (S1004A, vimentin, α‐smooth muscle actin, CD45) markers.Results: Cultured melanocytes displayed mostly bipolar morphology with heterogeneous eumelanin density related to donor eye iris colour. Whole transcriptome findings showed enriched melanogenesis genes (high level TYR, PMEL, MITF, TYRP1; MLANA; low level DCT1, MC1R) but not genes reported for RPE (e.g. RPE65), pericytes (e.g. ACTA2) or fibroblasts (e.g. IGF2) confirming melanocyte culture quality. We also found expression for proposed Schwann cell precursors PLP1 and SOX10. Immunolabelling confirmed overall expression of melanogenesis proteins, and subpopulations expressing PLP1 and Sox10, in culture and in choroidal tissue.Conclusions: Melanocytes and stromal fibroblast are among the most numerous and complex heterogeneous populations within the choroid and our study provides further details on characteristics markers and topography. A possible role for Schwann cell precursors in renewing adult melanocyte populations is intriguing and continues to be investigated.

The human capsular bag model as a pre‐clinical evaluation tool for IOLs

Cataract affects the vision and well‐being of millions. At present the only means of treating cataract is through surgical intervention. Modern cataract surgery typically retains a ring of anterior lens capsule and the entire posterior capsule. This is known as a capsular bag, which can house an intraocular lens (IOL) implant. Initial surgical outcomes are excellent, but with time lens cells that remain following surgery undergo a wound‐healing response. In essence the trauma of surgery provokes cells to proliferate and migrate across the acellular capsular surfaces and in some case the IOL surface. Most importantly, cells ultimately grow within the visual axis, on the central posterior capsule, and undergo fibrotic events that are associated with transdifferentiation to myofibroblasts, matrix deposition and matrix contraction that can lead to a secondary loss of vision termed posterior capsule opacification (PCO). PCO affects a large proportion of patients and is one of the most common fibrotic conditions in the world. It is therefore of great importance to better understand this condition and thus strong experimental systems are required for this purpose. To this end cataract surgery can be performed on human donor eyes within a laboratory setting to provide a biological product that is configured in the same manner as in the patient. This is known as the human capsular bag model. Using this clinically relevant model it has been possible to tease our key regulatory molecules driving human lens growth and fibrosis. While the human capsular bag model has been a useful tool to identify therapeutic targets or test putative therapeutics no pharmacological treatment is currently in clinical use. As a result, the intraocular lens is currently the major tool employed to manage PCO progression. Characteristics required for an IOL include strong optical performance, stability within the lens capsular bag and anti‐PCO properties. To meet international standards for IOL manufacture a number of tests are performed to predict IOL behaviour when implanted. However, ISO guided tests take place in a non‐biological setting and are limited to assessing purely physical characteristics. Therefore, understanding how the IOL interacts with the capsular bag and how wound‐healing responses could influence IOL position and stability is an area of great importance. The human capsular bag model can address this shortfall in the IOL development pipeline to relate design features to IOL stability and anti‐PCO performance in a clinically relevant setting. The presentation will highlight the evolution of the model as a tool for IOL testing and development.

Soemmerring's rings developed in‐vivo can present internal transparent areas

Purpose: To test whether any part of Soemmerring's rings developed, in‐vivo, in adults could be transparent and how this transparency relates to morphological factors.Methods: Sixteen Soemmerring's rings were extracted from donor eye globes. After frontal imaging, they were thickly sectioned sagittally in order to analyse the degrees of transparency of the cross‐section both at the periphery and center. All samples were also histologically analysed using alpha smooth muscle actin, Vimentin, wheat germ agglutinin and DAPI in order to correlate transparency to certain morphological factors.Results: When viewed frontally within the dark field, all Soemmerring's ring samples were apparently opaque. However, after sectioning, 20 of the 30 samples were more transparent in the center than at the periphery. Of the morphological factors that we analysed, only lens fibre organization at the bow region and an increased area of mature lens fibre cells had a significant relation to the degree of transparency at the center of cross‐sections.Conclusions: Soemmerring's rings can maintain the ability to produce organized and transparent areas of lens cells.

Imaging the Retinal Vascular Mural Cells In Vivo: Elucidating the Timeline of Their Loss in Diabetic Retinopathy.

Vascular mural cells (VMCs) are integral components of the retinal vasculature with critical homeostatic functions such as maintaining the inner blood-retinal barrier and vascular tone, as well as supporting the endothelial cells. Histopathologic donor eye studies have shown widespread loss of pericytes and smooth muscle cells, the 2 main VMC types, suggesting these cells are critical to the pathogenesis of diabetic retinopathy (DR). There remain, however, critical gaps in our knowledge regarding the timeline of VMC demise in human DR. In this study, we address this gap using adaptive optics scanning laser ophthalmoscopy to quantify retinal VMC density in eyes with no retinal disease (healthy), subjects with diabetes without diabetic retinopathy, and those with clinical DR and diabetic macular edema. We also used optical coherence tomography angiography to quantify capillary density of the superficial and deep capillary plexuses in these eyes. Our results indicate significant VMC loss in retinal arterioles before the appearance of classic clinical signs of DR (diabetes without diabetic retinopathy versus healthy, 5.0±2.0 versus 6.5±2.0 smooth muscle cells per 100 µm; P<0.05), while a significant reduction in capillary VMC density (5.1±2.3 in diabetic macular edema versus 14.9±6.0 pericytes per 100 µm in diabetes without diabetic retinopathy; P=0.01) and capillary density (superficial capillary plexus vessel density, 37.6±3.8 in diabetic macular edema versus 45.5±2.4 in diabetes without diabetic retinopathy; P<0.0001) is associated with more advanced stages of clinical DR, particularly diabetic macular edema. Our results offer a new framework for understanding the pathophysiologic course of VMC compromise in DR, which may facilitate the development and monitoring of therapeutic strategies aimed at VMC preservation and potentially the prevention of clinical DR and its associated morbidity. Imaging retinal VMCs provides an unparalleled opportunity to visualize these cells in vivo and may have wider implications in a range of diseases where these cells are disrupted.

Effect of Anterior Chamber Air on Central Corneal Thickness in Human Donor Eyes.

The purpose of this study was to describe the effects of intracameral air on corneal edema. A laboratory investigation was performed on human donor corneas. Baseline pachymetry measurements through anterior segment optical coherence tomography and endothelial cell density were obtained for all corneas. Each pair of corneas was separated and randomly assigned to undergo air injection or Optisol-GS into a BIONIKO artificial anterior chamber for 5 minutes at physiologic intraocular pressure confirmed by digital palpation. Photographs were obtained immediately on connection of the cornea to the artificial anterior chamber and on completion of the 5 minutes of treatment, with anterior chamber air being exchanged for Optisol-GS. Pretreatment and posttreatment photographs were obtained. Immediately after treatment, pachymetry was again obtained on all corneas. Pachymetry data underwent statistical analysis. Corneal pachymetry improved from 690.5 ± 126.6 to 576.1 ± 87.2 μm, yielding a 114.4 ± 50.4 μm improvement of pachymetry in the group with air injected into the anterior chamber. This was a significant improvement of pachymetry when compared with the group with Optisol-GS injected into the anterior chamber, which showed an improvement from 662.3 ± 126.5 to 613.5 ± 108.0 μm, yielding an improvement of 48.8 ± 34.3 μm. Injection of air into the anterior chamber leads to a significant decrease in corneal pachymetry. We thereby propose that injecting air intracamerally is an effective intraoperative intervention when visualization is negatively affected by corneal edema.

Effects of donor-specific microvascular anatomy on hemodynamic perfusion in human choriocapillaris

Evidence from histopathology and clinical imaging suggest that choroidal anatomy and hemodynamic perfusion are among the earliest changes in retinal diseases such as age-related macular degeneration (AMD). However, how inner choroidal anatomy affects hemodynamic perfusion is not well understood. Therefore, we sought to understand the influences of choroidal microvascular architecture on the spatial distribution of hemodynamic parameters in choriocapillaris from human donor eyes using image-based computational hemodynamic (ICH) simulations. We subjected image-based inner choroid reconstructions from eight human donor eyes to ICH simulation using a kinetic-based volumetric lattice Boltzmann method to compute hemodynamic distributions of velocity, pressure, and endothelial shear stress. Here, we demonstrate that anatomic parameters, including arteriolar and venular arrangements and intercapillary pillar density and distribution exert profound influences on inner choroidal hemodynamic characteristics. Reductions in capillary, arteriolar, and venular density not only reduce the overall blood velocity within choriocapillaris, but also substantially increase its spatial heterogeneity. These first-ever findings improve understanding of how choroidal anatomy affects hemodynamics and may contribute to pathogenesis of retinal diseases such as AMD.

Intraoperative prevention of postoperative ammetropies by intrastromal ring implantation

The experiment was carried out on 6 isolated donor (cadaver) eyes, penetrating keratoplasty was performed with intrastromal ring implantation according to the proposed method.When modeling the technology we proposed, we obtained patterns identical to those given, we observed a complete cut of the cornea without the formation of tissue bridges and adhesions. The developed new femtosecond laser cutting patterns made it possible to implant the intrastromal ring into the residual corneal stroma of the recipient without the need for additional fixation.