Electrophysiological findings in long-term type 1 diabetes patients without diabetic retinopathy using different ERG recording systems.

To assess full-field electroretinogram findings in long-term type 1 diabetes patients without diabetic retinopathy. Prospective study including 46 eyes of 23 patients with type 1 diabetes and 46 age-matched healthy eyes evaluated by the RETI-port/scan21 and the portable system RETeval following ISCEV guidelines. The average duration of diabetes was 28.88 ± 8.04 years. In scotopic conditions, using the RETI-port/scan21, diabetic patients showed an increase in b-wave implicit time (IT) (p = 0.017) with the lowest stimuli; a diminished b-wave amplitude (p = 0.005) in the mixed response, an increased IT (p = 0.004) with the high-intensity stimuli and an OP2 increased IT (p = 0.008) and decreased amplitude (p = 0.002). Under photopic conditions, b-wave amplitude was lower (p < 0.001) and 30-Hz flicker response was diminished (p = 0.021). Using the RETeval, in scotopic conditions, diabetic patients showed a reduction in the rod b-wave amplitude (p = 0.009), an increase in a-wave IT with the 280 Td.s stimulus (p = 0.005). OP2 had an increased IT and diminished amplitude (p = 0.003 and p = 0.002 respectively). 16 Td.s flicker showed an increased IT (p = 0.008) and diminished amplitude (p = 0.048). Despite variations in values between both systems, nearly all results displayed positive correlations. Long-term type 1 diabetes patients without diabetic retinopathy exhibit alterations in scotopic conditions, as evidenced by both conventional and portable electroretinogram devices. These findings suggest a modified retinal function, particularly in rod-driven pathways, even in the absence of vascular signs.

Evaluation of Macular Thickness Changes after Uncomplicated Phacoemulsification Surgery in Healthy Subjects and Diabetic Patients without Retinopathy by Spectral Domain OCT.

PURPOSE: To assess differences in the evolution of macular thickness after uncomplicated phacoemulsification surgery between non-diabetic subjects and patients with diabetes mellitus (DM) without diabetic retinopathy (DR), using Spectral Domain OCT (SD-OCT). METHODS: We performed a unicentric prospective study including one hundred and thirty-one eyes of 70 patients divided into two groups-34 well-controlled DM patients without DR and 36 non-diabetic subjects-who underwent phacoemulsification for cataract surgery. Eyes that developed pseudophakic cystoid macular edema (PCME) were excluded from the study, leaving us with 64 patients. Macular thickness was analyzed using Cirrus HD-OCT (Macular Cube 512 × 128 protocol) preoperatively and on postoperative days 7, 30, 90, and 180. For cases with information available for both eyes, one eye was randomly selected for analysis. RESULTS: A total of 64 eyes from 64 patients were analyzed in this study. The mean value of HbA1c in the diabetic group was 7%. After uncomplicated cataract surgery, patients showed no increase of the foveal, parafoveal, and perifoveal retinal thickness on postoperative day 7. However, thickness values increased on days 30, 90, and 180 after surgery in both groups, and peak at 90 days. There was no difference in macular thickness before or after surgery between DM and non-diabetic patients (p = 0.540). CONCLUSION: Macular thickness increases up to 6 months after uncomplicated cataract surgery in both DM patients without DR and non-diabetic subjects, with no differences between increases in both groups.

Long-Term Follow-Up of Macular Perfusion Evaluated by Optical Coherence Tomography Angiography after Rhegmatogenous Retinal Detachment Surgery.

BACKGROUND: The goal of this study was to investigate macular microvascular changes using optical coherence tomography angiography (OCTA) at one year after successful rhegmatogenous retinal detachment (RRD) surgery. METHODS: We performed a cross-section study including RRD treated by pars plana vitrectomy (PPV) with or without scleral buckling and SF6 tamponade. After 12 months, DRI-Triton SS-OCTA was performed. Superficial and deep retinal capillary plexuses (SCP and DCP), choriocapillaris (CC) vessel density (VD), and foveal avascular zone (FAZ) morphology were analyzed. Results were compared with the unaffected contralateral eye. RESULTS: Sixty eyes were included. We observed an increase in VD in the central area of both the SCP and DCP in macula-off eyes treated with PPV + SB and in the SCP of macula-off eyes treated with PPV. Macula-off eyes had a diminished VD for both plexuses in the superior quadrant and in the SCP inferior quadrant in those treated with PPV + SB. The CC flow was diminished in the temporal quadrant of macular-off eyes treated with PPV + SB. Healthy eyes presented higher diameter values than macula-off eyes treated with PPV + SB. FAZ horizontal and vertical diameters were smaller in patients with macula-off RRD vs. macula-on RRD and control groups. CONCLUSION: Macular vascularity remains almost unchanged one year after successful RRD surgery, irrespective of the surgical technique or prior macular status.

Systemic epigallocatechin gallate protects against retinal degeneration and hepatic oxidative stress in the P23H-1 rat.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders that lead to photoreceptor loss. RP has been reported to be related to oxidative stress, autophagy, and inflammation. (-)-Epigallocatechin gallate (EGCG), the most abundant catechin-based flavonoid in green tea leaves, has significant antioxidant, anti-carcinogenic, antimicrobial, and neuroprotective properties. EGCG, given its low molecular weight and hydrophilic properties, can cross the blood-retinal barrier and is able to reach different ocular tissues such as the lens, cornea, and retina. EGCG has been shown to provide retinal protection against ischemia; sodium nitroprusside-, N-methyl-D-aspartate-, lipopolysaccharide-, light-, sodium iodate-, or H2O2-induced damage and diabetic retinopathy. This suggests that systemic EGCG administration has the potential to protect against retinal degenerative or neurodegenerative diseases such as RP. The aim of this work was to investigate whether EGCG can protect against RP progression in the animal P23H line 1, the model of RP. Albino P23H rats were crossed with pigmented Long Evans rats to produce offspring exhibiting the clinical features of RP. Pigmented P23H rats were treated via intraperitoneal injection with saline or EGCG at a dose of 25 mg/kg every week from P100 to P160 and then compared to wild-type Long Evans rats. Rats treated with EGCG showed better visual and retinal electrical function with increased contrast sensitivity and b-wave values compared with those observed in P23H rats treated with vehicle. EGCG reduced lipid peroxidation and increased total antioxidant capacity and catalase and superoxide dismutase activities. No differences were observed in visual acuity, nitrate levels, nitrite levels or glutathione S-transferase activity. In conclusion, EGCG not only reduced the loss of visual function in P23H rats but also improved the levels of antioxidant enzymes and reduced oxidative damage. This study was approved by the Institutional Animal Care and Use Committee (CEICA) from the University of Zaragoza under project license PI12/14 on July 11, 2014.

Microperimetry-Assessed Functional Alterations and OCT-Changes in Patients after Retinal Detachment Surgery Using Pars Plana Vitrectomy and SF6 Tamponade.

BACKGROUND: We study the retinal function measured by macular integrity assessment microperimetry (MAIA) and structural changes assessed by scanning swept source optical coherence tomography (SS-OCT) between healthy individuals and patients undergoing pars plana vitrectomy (PPV) after rhegmatogenous retinal detachment (RRD). METHODS: Cross-sectional study. Early Treatment Diabetic Retinopathy Study (ETDRS) grids were measured by SS-OCT and compared with the MAIA parameters. RESULTS: Thirty-eight eyes with RRD (19 macula-on and 19 macula-off) were compared with 113 healthy eyes. The retinal sensitivity and average total threshold were reduced in all sectors in the RRD group; macular integrity index was increased. Macular thicknesses in total retina and ganglion cell layer (GCL)++ protocols were higher in the RRD group in nasal outer (NO) and central (C) sectors and only in C sector for GCL+ protocol. Thicknesses were lower in total retina, GCL++ protocols in the temporal outer (TO) sector and in the GCL+ protocol in NO sector. Best-corrected visual acuity (BCVA) correlated moderately with retinal sensitivity in all sectors and in just several sectors with time between the date of surgery and the test. The central nasal (CN) sector thickness and the average total threshold were higher in the macula-on subgroup. CONCLUSIONS: RRD and subsequent surgery results in functional and structural changes, especially in individuals with macular detachment.

Structural and functional findings in patients with moderate diabetic retinopathy.

PURPOSE: To evaluate structural and functional ocular changes in patients with type 2 diabetes mellitus (DM2) and moderate diabetic retinopathy (DR) without apparent diabetic macular edema (DME) assessed by optical coherence tomography (OCT) and microperimetry. METHODS: This was a single-center cross-sectional descriptive study for which 75 healthy controls and 48 DM2 patients with moderate DR were included after applying exclusion criteria (one eye per patient was included). All eyes underwent a complete ophthalmic examination (axial length, macular imaging with swept-source OCT, and MAIA microperimetry). Macular thicknesses, ganglion cell complex (GCC) thicknesses, and central retinal sensitivity were compared between groups, and the relationships between the OCT and microperimetry parameters were evaluated. RESULTS: Macular thickness was similar in both groups (242.17 ± 35.0 in the DM2 group vs 260.64 ± 73.9 in the control group). There was a diminution in the parafoveal area thickness in the DM2 group in the GCC complex. Retinal sensitivity was reduced in all sectors in the DM2 group. The central global value was 24.01 ± 5.7 in the DM2 group and 27.31 ± 2.7 in the control group (p < 0.001). Macular integrity was 80.89 ± 26.4 vs 64.70 ± 28.3 (p < 0.001) and total mean threshold was 23.90 ± 4.9 vs 26.48 ± 2.6 (p < 0.001) in the DM2 and control group, respectively. Moderate correlations were detected between the central sector of MAIA microperimetry and retina total central thickness (- 0.347; p = 0.0035). Age, visual acuity, and hemoglobin A1c levels also correlated with retinal sensitivity. CONCLUSION: Macular GCC thickness and central retinal sensitivity were reduced in patients with moderate DR without DME, suggesting the presence of macular neurodegeneration.

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Management of diabetic macular edema patients in clinical practice in Spain.

PURPOSE: Diabetic macular edema is the main cause of blindness in diabetic patients. Vascular endothelial growth factor is involved in diabetic macular edema pathogenesis. Vascular endothelial growth factor inhibitors are an important option in diabetic macular edema therapy. This survey investigates actual clinical practice in diabetic macular edema in Spain. METHODS: An expert advisory panel of 17 Spanish ophthalmologists developed a 30-item anonymous questionnaire about diagnosis, treatment, and follow-up in diabetic macular edema. A total of 137 ophthalmologists from 10 Spanish regions completed the questionnaire online. RESULTS: Almost all of the respondents (99.3%) record the measured visual acuity and perform biomicroscopic anterior (94.9%) and posterior (91.2%) segment examinations. Similarly, 100% of responding ophthalmologists always/almost always or frequently perform optical coherence tomography. Most respondents (65%) always/almost always or frequently perform a retinography. More than 50% rarely perform fluorescein angiography. Nearly, all (96.4%) of the specialists responded that, in center-involved diabetic macular edema, the first treatment is an anti-vascular endothelial growth factor drug. For corticosteroids, the first choice of most respondents (91.2%) was the dexamethasone implant. In the follow-up, almost all (96.4%) specialists record the measured visual acuity and most also perform biomicroscopic anterior (82.5%) and posterior (83.2%) segment examination. CONCLUSION: This survey shows the actual clinical practice in diabetic macular edema in Spain, finding that anti-vascular endothelial growth factor therapy is frequently used, and that diagnosis, treatments, and follow-up examinations used by specialists are homogeneous and according to diabetic macular edema guidelines.

Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors.

Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration.

Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells.

Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC-OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents. Stem Cells 2016;34:2625-2634.

Evaluation of Total Corneal Thickness and Corneal Layers With Spectral-Domain Optical Coherence Tomography.

PURPOSE: To evaluate total corneal thickness and corneal layers in healthy young adults using spectral-domain optical coherence tomography and to describe its repeatability and reproducibility. METHODS: Eighty-six eyes from 86 healthy volunteers were prospectively and consecutively enrolled. Manual measurements of central corneal thickness (CCT) and central thickness of epithelium, Bowman's layer, stroma, and the Descemet-endothelium complex were performed using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). To assess the reliability of the repeated measurements, intraclass correlation coefficients and coefficients of variation were used. RESULTS: Mean CCT, epithelium, Bowman's layer, stroma, and Descemet-endothelium values were 555.50 ± 29.64, 54.60 ± 4.25, 16.70 ± 1.73, 467.51 ± 28.91, and 16.74 ± 1.66 µm, respectively. The intraclass correlation coefficients ranged from 0.746 (Bowman's layer) to 0.999 (CCT and stroma) and from 0.483 (Bowman's layer) to 0.995 (CCT) and 0.998 (stroma) for intraobserver repeatability and interobserver reproducibility, respectively. The measurements showed coefficients of variation lower than 11% in all cases. CONCLUSIONS: This study establishes a normal database for corneal thickness and all its layers in healthy young adults with spectral-domain optical coherence tomography. This device exhibited a high degree of intraobserver repeatability and interobserver reproducibility for all regions except Bowman's layer.

Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases.

Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects, increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they all frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller cells and microglia, because their response to injury is decisive for maintaining the health of the retina or its degeneration. Several therapeutic approaches have been developed to preserve retinal function or restore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy, cell transplantation or artificial devices should be applied at the appropriate stage of retinal degeneration to obtain successful results. This review provides an overview of the common and distinctive features of retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused by the cellular response to damage, in order to establish appropriate treatments for these pathologies.

Induced pluripotent stem cells as custom therapeutics for retinal repair: progress and rationale.

Human pluripotent stem cells have made a remarkable impact on science, technology and medicine by providing a potentially unlimited source of human cells for basic research and clinical applications. In recent years, knowledge gained from the study of human embryonic stem cells and mammalian somatic cell reprogramming has led to the routine production of human induced pluripotent stem cells (hiPSCs) in laboratories worldwide. hiPSCs show promise for use in transplantation, high throughput drug screening, "disease-in-a-dish" modeling, disease gene discovery, and gene therapy testing. This review will focus on the first application, beginning with a discussion of methods for producing retinal lineage cells that are lost in inherited and acquired forms of retinal degenerative disease. The selection of appropriate hiPSC-derived donor cell type(s) for transplantation will be discussed, as will the caveats and prerequisite steps to formulating a clinical Good Manufacturing Practice (cGMP) product for clinical trials.

Safranal, a saffron constituent, attenuates retinal degeneration in P23H rats.

Saffron, an extract from Crocus sativus, has been largely used in traditional medicine for its antiapoptotic and anticarcinogenic properties. In this work, we investigate the effects of safranal, a component of saffron stigmas, in attenuating retinal degeneration in the P23H rat model of autosomal dominant retinitis pigmentosa. We demonstrate that administration of safranal to homozygous P23H line-3 rats preserves both photoreceptor morphology and number. Electroretinographic recordings showed higher a- and b-wave amplitudes under both photopic and scotopic conditions in safranal-treated versus non-treated animals. Furthermore, the capillary network in safranal-treated animals was preserved, unlike that found in untreated animals. Our findings indicate that dietary supplementation with safranal slows photoreceptor cell degeneration and ameliorates the loss of retinal function and vascular network disruption in P23H rats. This work also suggests that safranal could be potentially useful to retard retinal degeneration in patients with retinitis pigmentosa.

Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine.

Choroidal and optic disk metastases of a laryngeal carcinoma.

PURPOSE: To report a case of laryngeal squamous carcinoma metastatic to the eye affecting the choroid and optic nerve. METHODS: A 58-year-old man complained of sudden decrease of visual acuity in his left eye on admission to the emergency room. One year previously, he had undergone a surgical removal of laryngeal carcinoma. Funduscopic examination detected a choroidal mass in the macular area. RESULTS: After 10 days, the patient complained of pain and a diminished visual acuity and presented an exudative retinal detachment and optic nerve infiltration. CONCLUSION: Metastatic tumors are the most common intraocular malignancies, and the choroid is by far the most frequent location for the intraocular metastases. Metastases from a laryngeal carcinoma are quite unusual. Lesions affecting both choroid and optic nerve are extremely rare.

Changes in the inner and outer retinal layers after acute increase of the intraocular pressure in adult albino Swiss mice.

In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry was investigated at intervals ranging 3-14 weeks. Ocular hypertension (OHT) was induced by cauterizing the vessels draining the anterior part of the mice eye, as recently reported (Salinas-Navarro et al., 2009a). Electroretinographic (ERG) responses were recorded simultaneously from both eyes and compared each other prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processed at 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processed for immunocytochemistry using antibodies against recoverin, gamma-transducin, Protein Kinase C-alpha (PKC-alpha), calbindin or synaptophysin. The synaptic ribbons were identified using an antibody against the protein bassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photocoagulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocular pressure to approximately over twice its baseline by 24 h that was maintained for approximately five days reaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-wave and scotopic threshold response (STR) amplitudes, when compared to their contralateral fellow eye, reduced to 62%, 52% and 23% at 12 weeks after lasering. Three weeks after lasering, immunostaining with recoverin and transducin antibodies could not document any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells had lost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cell processes were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodies showed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontal cells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitative studies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 microm, and in the thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increased intraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses and in changes of the inner and outer retinal circuitries.