Early Choriocapillaris Loss in a Porcine Model of RPE Cell Debridement Precedes Pathology That Simulates Advanced Macular Degeneration.

Purpose: No large-mammal surgical models exist for geographic atrophy (GA), choroidal neovascularization (CNV), and pachychoroidal vascular remodeling. Our goal was to develop a porcine RPE debridement model of advanced macular degeneration to study photoreceptor cell loss and choroidal remodeling. Methods: Seven 2-month-old female domestic pigs were used for this study. After 25G vitrectomy, the area centralis was detached via subretinal bleb. A nitinol wire (Finesse Flex Loop) was used to debride RPE cells across a 3- to 5-mm diameter region. Fluid-air exchange was performed, and 20% SF6 gas injected. Animals underwent fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT-angiography (OCTA) at 2 weeks, 1 month, 2 months, 3 months, and 6 months postoperatively. Retinal histology was obtained at euthanasia, 2 months (n = 3), 3 months (n = 2), or 6 months (n = 2) after surgery. Results: RPE debridement resulted in GA with rapid loss of choriocapillaris, progressive loss of photoreceptors, and pachychoroidal changes in Sattler's and Haller's layers in all seven eyes undergoing debridement within 2 months. OCT and histological findings included subretinal disciform scar with overlying outer retinal atrophy; outer retinal tubulations and subretinal hyper-reflective material. OCTA revealed type 2 CNV (n = 4) at the edges of the debridement zone by 2 months, but there was no significant exudation noted at any time point. Conclusions: Surgical debridement of the RPE results in GA, CNV, and pachychoroid and reproduced all forms of advanced macular degeneration. This surgical model may be useful in examining the role of RPE and other cell replacement in treating advanced macular disease.

Methodological Approach to Improve Surgical Outcomes of a Pig Subretinal Implantation Model.

Purpose: To improve outcomes for subretinal implantation surgery in pigs. Methods: Analysis of variables affecting the success of subretinal implantation surgery was performed on videos of 37 surgeries. Ex vivo experiments were conducted to measure intraocular pressure (IOP) and test various prototyped implanters for effectiveness at maintaining IOP. Results: A video analysis revealed a prolonged sclerotomy open time owing to a combination of uncontrolled bleeding and excessive fluid outflow often resulting in retinal prolapse. Precauterization of the choroid before full-thickness sclerotomy (n = 10) resulted in a reduced incidence of uncontrolled bleeding from 39.1% (9/23) versus 0% (0/10) (P = 0.005) and improved implantation success from 73% to 90%. An ex vivo analysis of the IOP revealed a mean decrease in the IOP from 30.2 ± 3.0 mm Hg to 5.0 ± 2.1 mm Hg after a fully penetrating sclerotomy. To address this situation, we produced a series of plugs that integrated with a custom implant insertion device to seal the sclerotomy during implantation. The use of the plugs was cumbersome, however, and so we opted instead to increase the width of the inserter tip to fill the open sclerotomy. This improved device restored and maintained IOP during implantation (27.1 ± 1.9 mm Hg). Combined with precauterization the improved inserter resulted in 100% successful implantation (n = 4). Conclusions: For subretinal implantation in pigs, a modified procedure to precauterize the choroid before sclerotomy combined with an instrument that better fills the scleral opening decreases bleeding, hypotony, and open sclerotomy time, improving the success rate. Translational Relevance: Better management of IOP and bleeding from a sclerotomy will improve implant-based therapies.

Alteration of fibrin hydrogel gelation and degradation kinetics through addition of azo dyes.

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength.

Fibrin hydrogels are safe, degradable scaffolds for sub-retinal implantation.

Retinal pigment epithelium (RPE) transplantation for the treatment of macular degeneration has been studied for over 30 years. Human clinical trials have demonstrated that RPE monolayers exhibit improved cellular engraftment and survival compared to single cell suspensions. The use of a scaffold facilitates implantation of a flat, wrinkle-free, precisely placed monolayer. Scaffolds currently being investigated in human clinical trials are non-degradable which results in the introduction of a chronic foreign body. To improve RPE transplant technology, a degradable scaffold would be desirable. Using human fibrin, we have generated scaffolds that support the growth of an RPE monolayer in vitro. To determine whether these scaffolds are degraded in vivo, we developed a surgical approach that delivers a fibrin hydrogel implant to the sub-retinal space of the pig eye and determined whether and how fast they degraded. Using standard ophthalmic imaging techniques, the fibrin scaffolds were completely degraded by postoperative week 8 in 5 of 6 animals. Postmortem histologic analysis confirmed the absence of the scaffold from the subretinal space at 8 weeks, and demonstrated the reattachment of the neurosensory retina and a normal RPE-photoreceptor interface. When mechanical debridement of a region of native RPE was performed during implantation surgery degradation was accelerated and scaffolds were undetectable by 4 weeks. These data represent the first in situ demonstration of a fully biodegradable scaffold for use in the implantation of RPE and other cell types for treatment of macular degeneration and other retinal degenerative diseases.

Perfusion Bioreactor Culture of Bone Marrow Stromal Cells Enhances Cranial Defect Regeneration.

BACKGROUND: Cell-seeded biomaterial scaffolds have been proposed as a future option for reconstruction of bone tissue. The ability to generate larger, functional volumes of bone has been a challenge that may be addressed through the use of perfusion bioreactors. In this study, the authors investigated use of a tubular perfusion bioreactor system for the growth and differentiation of bone marrow stromal (mesenchymal stem) cells seeded onto fibrin, a highly angiogenic biomaterial. METHODS: Cells were encapsulated within fibrin beads and cultured either within a tubular perfusion bioreactor system or statically for up to 14 days. Scaffolds were analyzed for osteogenic differentiation. A rodent cranial defect model (8-mm diameter) was used to assess the bone regeneration of scaffolds cultured in the bioreactor, statically, or used immediately after formation. Immunohistochemistry was used to visualize CD31 vessel density. Micro-computed tomographic imaging was used to visualize mineral formation within the defect volume. RESULTS: Tubular perfusion bioreactor system-cultured samples showed significantly greater osteodifferentiation, indicated by an increase in VEGF expression and mineral deposition, compared with statically cultured samples. Increased expression of OPN, RUNX2, VEGF, and CD90 was seen over time in both culture methods. After implantation, bioreactor samples exhibited greater bone formation and vessel density compared with all other groups. Analysis of micro-computed tomographic images showed full union formation through the greatest diameter of the defect in all bioreactor samples and the highest levels of mineralized volume after 8 weeks. CONCLUSION: Mesenchymal stem cells encapsulated in fibrin beads and cultured in the tubular perfusion bioreactor system resulted in increased vascularization and mineralized tissue formation in vivo relative to static culture.

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions.

Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three-dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two-dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1-60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin-based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. Stem Cells Translational Medicine 2019;8:512-521.

Mutant Best1 Expression and Impaired Phagocytosis in an iPSC Model of Autosomal Recessive Bestrophinopathy.

Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents. After differentiation to retinal pigment epithelial (iPSC-RPE) cells, both BEST1 mRNA and Best1 protein expression were compared to controls. BEST1 mRNA expression levels, determined by quantitative PCR, were similar in ARB iPSC-RPE, parental cells, and genetically unrelated controls. Western blotting revealed that CRALBP and RPE65 were expressed within the range delineated by unrelated controls in iPSC-RPE from the ARB donor and her parents. Best1 protein was detected in different clones of ARB iPSC-RPE, but at reduced levels compared to all controls. When tested for the ability to phagocytose photoreceptor outer segments, ARB iPSC-RPE exhibited impaired internalization. These data suggest that impaired phagocytosis is a trait common to the bestrophinopathies. Furthermore, ARB is not universally the result of NMD and ARB, in this patient, is not due to the absence of Best1.

Fibrin hydrogels as a xenofree and rapidly degradable support for transplantation of retinal pigment epithelium monolayers.

Recent phase 1 trials of embryonic stem cell and induced pluripotent stem cell (iPSCs) derived RPE transplants for the treatment of macular degeneration have demonstrated the relative safety of this process. However, there is concern over clumping, thickening, folding, and wrinkling of the transplanted RPE. To deliver a flat RPE monolayer, current phase 1 trials are testing synthetic substrates for RPE transplantation. These substrates, however, cause localized inflammation and fibrosis in animal models due to long degradation times. Here we describe the use of thin fibrin hydrogels as a support material for the transplantation of RPE. Fibrin was formed into a mechanically rigid support that allow for easy manipulation with standard surgical instruments. Using fibrinolytic enzymes, fibrin hydrogels were degraded on the scale of hours. The rate of degradation could be controlled by varying the fibrinolytic enzyme concentration used. RPE cells degraded fibrin spontaneously. To preserve the fibrin support during differentiation of iPSCs to RPE, media was supplemented with the protease inhibitor aprotinin. iPSC-RPE on fibrin gels remained viable, generated monolayers with characteristic cobblestone appearance and dark pigmentation, and expressed mRNA and protein markers characteristic of RPE in the eye. Following differentiation of the cells, addition of fibrinolytic enzymes fully and rapidly degraded the fibrin support leaving behind an intact, viable iPSC-RPE monolayer. In conclusion, human fibrin hydrogels provide a xeno-free support on which iPSCs can be differentiated to RPE cells for transplant which can be rapidly degraded under controlled conditions using fibrinolytic enzymes without adverse effects to the cells. STATEMENT OF SIGNIFICANCE: Stem cell-derived retinal pigment epithelial (RPE) cell transplantation is currently in phase 1 clinical trials for macular degeneration (MD). A major obstacle in these studies is delivering the RPE as a living, flat sheets without leaving behind foreign materials in the retina. Here we investigate the suitability of using hydrogels made from human blood-derived proteins for RPE transplant. Our data shows that these fibrin hydrogels are rigid enough for use in surgery, support growth of stem cell-derived RPE, and are easily degraded within hours without damage to the RPE sheet. These fibrin hydrogels offer a promising solution to transplant RPE for patients with MD.

A Model to Study Thermal Energy Delivery to the Choroid: A Comparison of Surgical Devices.

PURPOSE: We measure and compare surgical devices using an ex vivo, temperature-controlled, choroidal incision model during thermal energy transfer with a high-resolution infrared camera. METHODS: Ex vivo porcine choroidal tissue specimens (n = 516) were isolated and placed on a temperature-regulated (37°C) perfusion platform. We tested the pulsed electron avalanche knife (PEAK), micropulse laser (MpL), continuous laser (CL), and bipolar cautery (BpC) at three energy settings (11 [low], 45 [medium], and 134 [high] mJ/mm). Each device was clamped to a stationary mechanical arm. Movement of tissue specimens beneath the surgical device was achieved using a stepping motor-driven x-y table. An infrared video camera measured orthogonal temperature variation in the surrounding tissue. RESULTS: Increased power resulted in greater lateral thermal spread using all modalities (P < 0.001). Mean (standard deviation) lateral thermal spread at low energy was smallest for the MpL at 0.0 (0.01) mm (P < 0.001), whereas BpC had the least collateral tissue damage at medium and high energies (0.02 [0.08] and 0.34 [0.22] mm, respectively; P < 0.001). Fluidics of the ex vivo system may limit thermal spread. The PEAK had the greatest thermal spread across all energy groups (P < 0.001), with clinically relevant variation between disposable blades. CONCLUSIONS: Our ex vivo model enabled direct comparison of threshold thermal tissue injury across four devices. MpL and BpC showed the least thermal damage. PEAK had a higher variation in energy delivery, but also has the advantage of more effective tissue cutting. TRANSLATIONAL RELEVANCE: Our ex vivo surgical device analysis provides thermal tissue injury predictions for choroidal surgery.

Differential Intraocular Pressure Measurements by Tonometry and Direct Cannulation After Treatment with Soluble Adenylyl Cyclase Inhibitors.

PURPOSE: To validate the increase in intraocular pressure (IOP) caused by soluble adenylyl cyclase (sAC) inhibitors and determine reasons behind variation in IOP measurements performed by tonometry. METHODS: C57BL/6J mice were administered DMSO solubilized sAC inhibitors (KH7 or LRE-1) by intraperitoneal injection. Two hours post-treatment, mice were anesthetized with avertin or ketamine/xylazine/acepromazine (KXA). IOP was measured by a rebound tonometer or direct cannulation of the anterior chamber. Spectral-domain optical coherence tomography was used to measure anterior chamber depth and corneal thickness in live mice. Outflow facility was measured in perfused, enucleated mouse eyes. RESULTS: Compared with DMSO controls, KH7 treatment caused an increased IOP in avertin- and KXA-anesthetized mice when measured by direct cannulation [avertin: 14.4 ± 2.1 mmHg vs. 11.1 ± 1.0 mmHg (P = 0.003); KXA: 14.4 ± 1.0 mmHg vs. 11.3 ± 0.8 mmHg (P < 0.001)] and tonometry [avertin: 10.8 ± 1.4 mmHg vs. 7.4 ± 0.6 mmHg (P < 0.001); KXA: 11.9 ± 0.9 mmHg vs. 10.3 ± 1.7 mmHg (P = 0.283)]. However, treatment with KH7 in nonanesthetized mice showed a significant decrease in IOP measured by tonometry and compared with DMSO-treated animals [13.1 ± 2.6 mmHg vs. 15.6 ± 0.5 mmHg (P = 0.003)]. Both KH7- and DMSO-treated groups anesthetized with avertin showed increased corneal thickness, whereas KH7-treated mice anesthetized with KXA exhibited a shallower anterior chamber compared with untreated mice. KH7 decreased outflow facility by 85.1% in nonanesthetized, enucleated eyes (P < 0.003). CONCLUSIONS: Systemically administered DMSO and anesthesia have significant effects on anterior chamber characteristics, resulting in altered IOP readings measured by tonometry. In the presence of DMSO and anesthesia, tonometry IOP readings should be confirmed with direct cannulation.

Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization.

Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating.

Falciform macular folds and chromosome 22q11.2: evidence in support of a locus for familial exudative vitreoretinopathy (FEVR).

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a genetic disease caused by abnormal retinal vascular development. New additional genetic loci for FEVR have recently been identified. Microduplication of 22q11.2 has been reported with a heterogeneous phenotype and microdeletion of 22q11.2 has been associated with FEVR. We describe a case of a girl with microduplication of 22q11.2 and falciform macular folds. MATERIALS AND METHODS: The infant and first-degree relatives were examined. A dilated fundus examination was performed. Genetic screening was done by chromosomal microarray analysis and confirmed by fluorescent in situ hybridization (FISH). RESULTS: Bilateral macular folds were found with temporal fibrosis in the proband. A chromosomal microarray revealed a 2.21 Mb microduplication of the 22q11.2 region. CONCLUSION: This is the first report to associate microduplication of 22q11.2 with macular folds, supporting the potential for a FEVR locus on chromosome 22q11.2. We encourage full ophthalmological examination for patients with microduplication of 22q11.2 to identify ocular associations.

Alginate-based strategies for therapeutic vascularization.

Therapeutic stimulation of vessel growth to improve tissue perfusion has shown promise in many regenerative medicine and tissue engineering applications. Alginate-based biomaterial systems have been investigated for growth factor and/or cell delivery as tools for modulating vessel assembly. Growth factor encapsulation allows for a sustained release of protein and protection from degradation. Implantation of growth factor-loaded alginate constructs typically shows an increase in capillary density but without vascular stabilization. Delivery of multiple factors may improve these outcomes. Cell delivery approaches focus on stimulating vascularization either via cell release of soluble factors, cell proliferation and incorporation into new vessels or alginate prevascularization prior to implantation. These methods have shown some promise but routine clinical application has not been achieved. In this review, current research on the application of alginate for therapeutic neovascularization is presented, shortcomings are addressed and the future direction of these systems discussed.

Soluble CD59 expressed from an adenovirus in vivo is a potent inhibitor of complement deposition on murine liver vascular endothelium.

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited.

TNFalpha is required for late BRB breakdown in diabetic retinopathy, and its inhibition prevents leukostasis and protects vessels and neurons from apoptosis.

PURPOSE: Blood-retinal barrier [BRB] breakdown, characteristic of diabetic retinopathy (DR), is believed to depend on inflammation and apoptosis. Retinal inflammation is almost completely suppressed in the absence of TNFα, which is also associated with apoptosis. This study was conducted to determine the role of TNFα in these diabetic complications. METHODS: Diabetes was induced with streptozotocin in Tnfa knockout (KO) mice, to provide a chemical model of diabetes, and Tnfa (KO) mice were crossed with Ins2(Akita) mice to generate a genetic model, with both models being devoid of TNFα. The BRB was assessed at 1, 1.5, 3, and 6 months. Leukostasis was assessed using FITC-conjugated ConA to label leukocytes. Apoptosis was assessed with TUNEL and activated caspase-3 staining. PECAM1 identified endothelial cells, and SMA identified pericytes. RESULTS: At 1 month of diabetes, the absence of TNFα had no effect on DR-associated BRB breakdown, even though it prevented retinal leukostasis, demonstrating that neither TNFα nor inflammation is essential for early BRB breakdown in DR in either model of diabetes. At 3 months of diabetes, BRB breakdown was significantly suppressed and at 6 months, it was completely prevented in the absence of TNFα in both models, showing that TNFα is essential for progressive BRB breakdown. DR-mediated apoptosis in the retina, which appears to involve endothelial cells, pericytes, and neurons, was inhibited in the absence of TNFα in both models. CONCLUSIONS: Although neither TNFα nor inflammation is necessary for early BRB breakdown in DR, TNFα is critical for later complications and would be a good therapeutic target for the prevention of the progressive BRB breakdown, retinal leukostasis, and apoptosis associated with DR.

Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP): 24-month experience with telemedicine screening.

PURPOSE: To report the 24-month experience of the Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP) telemedicine initiative. METHODS: Retrospective analysis of the SUNDROP archival data gathered between 1 December 2005 and 30 November 2007 to evaluate this diagnostic technology for ROP screening. One hundred and sixty consecutively enrolled infants meeting ROP examination criteria were screened with the RetCam II and evaluated by the SUNDROP reading centre at Stanford University. Nurses obtained five or six images in each eye. All patients also received a dilated examination within 1 week of discharge. Outcomes included treatment-warranted retinopathy of prematurity (TW-ROP) and anatomical outcomes. RESULTS: In the initial 24-month period, the SUNDROP telemedicine initiative has not missed any TW-ROP. A total of 160 infants (320 eyes) were imaged, resulting in 669 exams and 7556 images. Seven infants were identified with TW-ROP; six underwent laser photocoagulation and one regressed spontaneously. The sensitivity was 100%, with specificity of 99.4%. No patient progressed to retinal detachment or other adverse outcomes. CONCLUSION: The SUNDROP telemedicine screening initiative for ROP has been proven to have a high degree of sensitivity and specificity for the identification of treatment-warranted disease. All cases of treatment-warranted disease were captured. There were no adverse outcomes.

Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP): 18-month experience with telemedicine screening.

PURPOSE: To report the 18-month experience of the Stanford University Network for Diagnosis of Retinopathy of Prematurity (SUNDROP) telemedicine initiative. DESIGN: Retrospective analysis of the SUNDROP archival data between 1 December 2005 and 30 May 2007, evaluating this new diagnostic technology for ROP screening. PARTICIPANTS: All 97 consecutively enrolled infants in the SUNDROP network. METHODS: All patients were screened using the RetCam II, and evaluated by the SUNDROP reading center at Stanford University. Nurses were trained to obtain five images in each eye. All patients were screened by an ophthalmologist trained in diagnosing ROP within 1 week of discharge from the hospital. MAIN OUTCOME MEASURES: Outcomes included referral-warranted disease, need for treatment, and anatomic outcomes. Referral-warranted disease was defined as any Early Treatment Retinopathy of Prematurity Disease Type 2 or greater, threshold disease, any plus disease, and any stage 4 or higher disease. RESULTS: In the initial 18-month period, the SUNDROP telemedicine screening initiative has not missed any referral-warranted disease for ROP. A total of 97 infants (194 eyes) were enrolled, resulting in 443 unique examinations and 4,929 unique images. The mean birth weight of the infants was 1,186.9 grams, with a mean gestational age at birth of 28.9 weeks. Seven infants were identified with referral-warranted disease; six patients underwent laser photocoagulation and completely regressed. The one remaining patient regressed spontaneously, and did not require intervention. Calculated sensitivity and specificity was 100% and 98.9% respectively. No patient progressed to retinal detachment or other adverse outcome. Inadequate exposure, artifact, poor visualization of the periphery, and lack of a complete standardized image set in some patients were identified as areas requiring further assessment. CONCLUSIONS: The SUNDROP telemedicine screening initiative for ROP has proven to have a high degree of sensitivity and specificity for identification of referral-warranted disease. Training was easily implemented. All cases of referral-warranted disease were captured. There were no adverse outcomes.

High-resolution imaging with adaptive optics in patients with inherited retinal degeneration.

PURPOSE: To investigate macular photoreceptor structure in patients with inherited retinal degeneration using high-resolution images and to correlate the findings with clinical phenotypes and genetic mutations. METHODS: Adaptive optics scanning laser ophthalmoscopy (AOSLO) images of photoreceptors were obtained in 16 eyes: five with retinitis pigmentosa (RP), three with cone-rod dystrophy (CRD), and eight without retinal disease. A quadratic model was used to illustrate cone spacing as a function of retinal eccentricity. Cone spacing at 1 degrees eccentricity was compared with standard measures of central visual function, including best-corrected visual acuity (BCVA), foveal threshold, and multifocal electroretinogram (mfERG) amplitude and timing. Intervisit variations were studied in one patient with RP and one patient with CRD. Screening of candidate disease genes identified mutations in two patients, one with RP (a rhodopsin mutation) and the other with CRD (a novel RPGR-ORF15 mutation). RESULTS: Cone spacing values were significantly different from normal for patients with RP (P = 0.01) and CRD (P < 0.0001) and demonstrated a statistically significant correlation with foveal threshold (P = 0.0003), BCVA (P = 0.01), and mfERG amplitude (P = 0.008). Although many RP patients showed normal cone spacing within 1 degrees of fixation, cones could not be unambiguously identified in several retinal regions. Cone spacing increased in all CRD patients, even those with early disease. Little variation was observed in cone spacing measured during two sessions fewer than 8 days apart. CONCLUSIONS: AOSLO images can be used to study macular cones with high resolution in patients with retinal degeneration. The authors present the first report of cone structure in vivo in patients with mutations in rhodopsin and RPGR-ORF15 and show that macular cones display distinct characteristics, depending on the underlying disease. AOSLO imaging, therefore, can provide new insight into possible mechanisms of cone vision loss in patients with retinal degeneration.