LONGITUDINAL ELLIPSOID ZONE AND OUTER RETINAL INTEGRITY DYNAMICS AFTER EPIRETINAL MEMBRANE SURGERY.

PURPOSE: To quantify ellipsoid zone (EZ) changes in integrity after epiretinal membrane (ERM) surgery, correlate findings to visual acuity, and determine predictors for prognosis. METHODS: A post hoc analysis of eyes undergoing ERM surgery pooled from the prospective DISCOVER intraoperative optical coherence tomography study and eyes undergoing conventional ERM surgery without intraoperative optical coherence tomography. Quantitative EZ features were extracted using a multilayer machine learning enabled automated segmentation platform after image analyst review/correction for segmentation accuracy. Visual acuity and EZ integrity were quantitatively assessed and correlated before and after ERM surgery. Multiple linear regression was performed to assess preoperative visual acuity and EZ features as predictors for improvement in visual acuity or EZ integrity. RESULTS: There were 177 eyes from 177 subjects that underwent ERM surgery from the DISCOVER and conventional arms. Improvement in visual acuity and multiple EZ integrity features was noted after ERM surgery, including EZ partial attenuation and EZ-retinal pigment epithelium (RPE) volume (P < 0.05). A reduction in EZ partial attenuation and increase in EZ-RPE central subfield thickness (EZ-RPE CST) was significantly correlated with improved visual acuity after ERM surgery (P < 0.05). More robust EZ-RPE CST at baseline predicted visual acuity improvement after ERM peel in regression modeling (ß = 0.005, P < 0.05). CONCLUSIONS: Longitudinal assessment of EZ features demonstrates significant postoperative improvement in multiple EZ integrity metrics after ERM surgery. Improving EZ integrity was correlated to improving the visual acuity. Ellipsoid zone integrity and visual acuity were significant predictors in regression modeling and may have value in clinical prognostication.

In vivo evaluation of outer retinal function and structure after retrobulbar optic nerve crush by lateral orbitotomy in goats.

Large animal model of optic nerve crush (ONC) plays an important role in translating novel therapeutic strategies developed in rodent model to clinical application. Due to the poor accessibility of the optic nerve (ON) in humans and large animals, lateral orbitotomy is needed to expose the retrobulbar ON. This study was to explore the effects of ONC and ON exposure with lateral orbitotomy (sham surgery) on the outer retinal function and structure in goats by using standard flash electroretinogram (FERG) and spectral-domain optical coherence tomography (SD-OCT). We found that ONC led to a transient reduction in FERG amplitudes at 1 week post injury (wpi), which recovered gradually over 2 months afterwards. Sham surgery alone also caused a similar pattern of amplitude reduction in FERG, although not as significantly as ONC did. Transient outer retinal thickening following ONC occurred at 4 wpi (when progressive thinning of the ganglion cell complex began), peaked at 8 wpi, then recovered gradually at 12 wpi. In contrast, outer retinal thickness remained unchanged statistically 3 months after sham surgery. Fundus fluorescein angiography showed that neither ONC nor ON exposure with lateral orbitotomy significantly caused any significant delay or absence of central retinal vascular filling. In summary, ONC with lateral orbitotomy affects outer retinal function and structure transiently.

Structure and dynamics of photoreceptor sensory cilia.

The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structure and distribution of molecular components of these structures are still incompletely understood, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.

Light-induced modifications of the outer retinal hyperreflective layers on spectral-domain optical coherence tomography in humans: an experimental study.

PURPOSE: Numerous small hyperreflective dots (HRDs) can be seen within the hyporeflective layer between the ellipsoid zone (EZ) and the interdigitation zone (IZ) on C-scan spectral-domain optical coherence tomography (SD-OCT) with a yet unknown variation under light conditions. The aim of this study was to explore light-induced SD-OCT changes in these HRDs. METHODS: The study subjects were randomly assigned to two groups: Group 1 experienced a dark adaptation protocol followed by intense retinal photobleaching, while Group 2, serving as the control group, was exposed to constant ambient light without any variation. The number of HRDs was automatically counted. RESULTS: Twenty healthy volunteers were prospectively included. The number of HRDs differed significantly over time (p = 0.0013). They decreased in Group 1 after dark adaptation and retinal photobleaching before returning to baseline levels 30 min later; conversely, they remained relatively constant in Group 2 throughout the study (p < 0.001). Light-skinned subjects had less HRD than dark-skinned subjects. CONCLUSION: We observed light-induced modifications in the space between the EZ and the IZ. We hypothesize that the HRDs visible in this zone correspond to melanosomes that are mobilized during the light stimulation protocol. Larger studies are recommended to further evaluate and confirm light-induced SD-OCT changes under physiological and pathological conditions.

Live Imaging of Organelle Motility in RPE Flatmounts.

The retinal pigment epithelium (RPE) performs several functions that are crucial for normal retinal function and vision, including the daily phagocytosis of photoreceptor outer segment (POS) membranes. Defects in the motility and degradation of POS phagosomes may be associated with some inherited and age-related retinal degenerations. Given the apical to basal translocation of phagosomes during maturation and degradation, studies of the underlying mechanisms require analyses of the dynamics in 3-D. In this chapter, we report a method for investigating the 3-D motility of POS phagosomes and lysosomes, utilizing high-speed, spinning disk confocal microscopy of live RPE flatmounts.

The Role of the Prph2 C-Terminus in Outer Segment Morphogenesis.

Peripherin 2 (also known as RDS/Prph2) is localized to the rims of rod and cone outer segment (OS) discs. The C-terminus of Prph2 is a critical functional domain, but its exact role is still unknown. In this mini review, we describe work on the Prph2 C-terminus, highlighting its role as a regulator of protein trafficking, membrane curvature, ectosome secretion, and membrane fusion. Evidence supports a role for the Prph2 C-terminus in these processes and demonstrates that it is necessary for the initiation of OS morphogenesis.

A platform for assessing outer segment fate in primary human fetal RPE cultures.

The daily shedding and renewal of photoreceptor outer segments (OS) is critical for maintaining vision. This process relies on the efficient uptake, degradation, and sorting of shed OS material by the retinal pigment epithelium (RPE). Poor OS degradation has been linked to retinal degenerations such as Stargardt disease and may contribute to macular degeneration. While primary human fetal RPE cultures have emerged as a valuable model of in vivo human RPE function, surprisingly few studies have utilized the model for tracking the degradation and fate of OS components in the RPE. Here, we establish an improved platform for studying this topic by modifying existing protocols and creating new methods. Our human fetal culture model facilitates studies of RPE secretion in response to OS ingestion, preserves RPE differentiation and polarization during live-cell imaging of OS phagocytosis, and minimizes costs. We optimize Mer tyrosine kinase-dependent OS phagocytosis assays specifically in human fetal cultures and provide a simple and accurate method for measuring total OS consumption by the RPE. Finally, we utilize chemical transfection, dextran labeling, and immunocytochemistry to evaluate key players in OS degradation, including lysosomes and autophagy proteins. To facilitate quantification of autophagy vesicles, we develop customized image analysis macros in the Fiji/ImageJ software environment. These protocols will facilitate a broad range of studies in human fetal RPE cultures aimed at determining the ultimate fate of OS components after ingestion, a critical step in understanding the pathogenesis of numerous retinal diseases.

Outer Retinal Defects Represent a Normal Recovery Pathway Following Internal Limiting Membrane Peeling in Macular Hole Surgery.

BACKGROUND AND OBJECTIVE: To examine perioperative factors associated with the development of outer retinal defects (ORDs) following surgical repair of macular holes (MHs). PATIENTS AND METHODS: An institutional review board-approved, retrospective, interventional cohort study was conducted. Patients who underwent MH repair during a 5-year period were identified. Statistical analysis was conducted to detect significant perioperative associations to ORD development. RESULTS: One hundred twenty-four eyes were included, and 54% developed an ORD following surgery. These defects correlated with lower preoperative stage (P = .0057), preoperative phakia (P = .036), and lack of prior macular surgery (P = .0016). Patients in the ORD group had significantly better preoperative and postoperative visual acuity (P = .031 and P = .0004, respectively), but there was no difference in change in acuity from preoperatively to 3 months postoperatively when compared with control patients (P = 42). The majority (89%) of ORDs resolved by 24 months postoperatively. CONCLUSION: The development of ORDs appears to be correlated with several factors indicative of favorable overall eye health and less advanced pathology and may represent a normal state of recovery after MH repair with internal limiting membrane peeling. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e1-e8.].

Normal Variation of Photoreceptor Outer Segment Volume With Age, Gender, Refractive Error, and Vitreomacular Adhesion.

BACKGROUND AND OBJECTIVE: Thickness and volume changes of the photoreceptor outer segment (PROS) layer on spectral-domain optical coherence tomography (SD-OCT) images are associated with various disease states. However, there are limited data on normal anatomical variation. This study evaluates the correlation of PROS volume with age, gender, refractive error, and presence of vitreomacular adhesion (VMA) in healthy subjects. PATIENTS AND METHODS: SD-OCT scans of 68 normal eyes were analyzed. The ellipsoid zone and the apical retinal pigment epithelium boundary were segmented using an automated algorithm. The PROS volume was calculated as the region bounded by these two layers within a 6-mm diameter circle centered at the fovea. A general linear model was used for statistical analysis. RESULTS: PROS volume increased with age to a significant degree (P = .013). Gender, refractive error, and presence of VMA were insignificant factors. CONCLUSION: PROS volume, as measured on routine SD-OCT, increases with age in healthy subjects, after adjusting for gender, refractive error, and VMA. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:523-527.].

Oligomerization of Prph2 and Rom1 is essential for photoreceptor outer segment formation.

Mutations in peripherin 2 (PRPH2, also known as Rds), a tetraspanin protein found in photoreceptor outer segments (OSs), cause retinal degeneration ranging from rod-dominant retinitis pigmentosa (RP) to cone-dominant macular dystrophy (MD). Understanding why some Prph2 mutants affect rods while others affect cones remains a critical unanswered question. Prph2 is essential for OS structure and function and exhibits a very specific pattern of oligomerization with its homolog Rom1. Non-covalent Prph2/Rom1 homo- and hetero-tetramers assemble into higher-order covalently linked complexes held together by an intermolecular disulfide bond at Prph2-C150/Rom1-C153. Here we disrupt this crucial bond using a C150S-Prph2 knockin mouse line to study the role of Prph2 higher-order complex formation. We find that C150S-Prph2 traffics to the OS, interacts with Rom1 and forms non-covalent tetramers, but alone cannot support normal OS structure and function. However, C150S-Prph2 supports the initiation or elaboration of OS disc structures, and improves rod OS ultrastructure in the presence of wild-type (WT) Prph2 (i.e. Prph2C150S/+ versus Prph2+/-). Prph2C150S/+ animals exhibit haploinsufficiency in rods, but a dominant-negative phenotype in cones, suggesting cones have a different requirement for large Prph2 complexes than rods. Importantly, cone but not rod function can be improved by the addition of one Prph2Y141C allele, a mutation responsible for pattern dystrophy owing to the extra cysteine. Combined these findings show that covalently linked Prph2 complexes are essential for OS formation, but not for Prph2 targeting to the OS, and that cones are especially sensitive to having a broad distribution of Prph2 complex types (i.e. tetramers and large complexes).

The Role of the Microglial Cx3cr1 Pathway in the Postnatal Maturation of Retinal Photoreceptors.

Microglia are the resident immune cells of the CNS, and their response to infection, injury and disease is well documented. More recently, microglia have been shown to play a role in normal CNS development, with the fractalkine-Cx3cr1 signaling pathway of particular importance. This work describes the interaction between the light-sensitive photoreceptors and microglia during eye opening, a time of postnatal photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP ) led to an early retinal dysfunction soon after eye opening [postnatal day 17 (P17)] and cone photoreceptor loss (P30 onward) in mice of either sex. This dysfunction occurred at a time when fractalkine expression was predominantly outer retinal, when there was an increased microglial presence near the photoreceptor layer and increased microglial-cone photoreceptor contacts. Photoreceptor maturation and outer segment elongation was coincident with increased opsin photopigment expression in wild-type retina, while this was aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor cilium, a key structure that is important for outer segment elongation. This was confirmed with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, downregulated at eye opening (P14). While the overall cilium structure was unaffected, expression of Rpgr, Rpgrip1, and centrin were restricted to more proximal regions of the transitional zone. This study highlighted a novel role for microglia in postnatal neuronal development within the retina, with loss of fractalkine-Cx3cr1 signaling leading to an altered distribution of cilium proteins, failure of outer segment elongation and ultimately cone photoreceptor loss.SIGNIFICANCE STATEMENT Microglia are involved in CNS development and disease. This work highlights the role of microglia in postnatal development of the light-detecting photoreceptor neurons within the mouse retina. Loss of the microglial Cx3cr1 signaling pathway resulted in specific alterations in the cilium, a key structure in photoreceptor outer segment elongation. The distribution of key components of the cilium transitional zone, Rpgr, Rpgrip1, and centrin, were altered in retinae lacking Cx3cr1 with reduced outer segment length and cone photoreceptor death observed at later postnatal ages. This work identifies a novel role for microglia in the postnatal maturation of retinal photoreceptors.

The Retinol Binding Protein Receptor 2 (Rbpr2) is required for Photoreceptor Outer Segment Morphogenesis and Visual Function in Zebrafish.

Vitamin A (all-trans retinol) plays critical roles in mammalian development and vision. Since vitamin A is food-derived, tissue-specific uptake and storage mechanism are needed. In the eye, uptake of RBP4-retinol is mediated by the receptor Stra6, whereas the receptor mediating RBP4 binding and retinol transport into the liver has just recently been discovered. Here we examined the role of zebrafish retinol binding protein receptor 2 (Rbpr2) for RBP4-retinol uptake in developing embryos, using eye development and vision as sensitive readouts. In cultured cells, Rbpr2 localized to membranes and promoted RBP4-retinol uptake. In larvae, Rbpr2 expression was detected in developing intestinal enterocytes and liver hepatocytes. Two rbpr2 mutant zebrafish lines, each resulting in Rbpr2 deficiency, exhibit a small eye defect, and systemic malformations including hydrocephaly and cardiac edema, phenotypes associated with vitamin A deficiency. In the retina, Rbpr2 loss resulted in shorter photoreceptor outer segments, mislocalization and decrease in visual pigments, decreased expression of retinoic acid-responsive genes and photoreceptor cell loss, overall leading to a reduction of visual function. Together, these results demonstrate that Rbpr2-mediated RBP4-retinol uptake in developing liver and intestine is necessary to provide sufficient substrate for ocular retinoid production required for photoreceptor cell maintenance and visual function.

Photoreceptor assessment using adaptive optics in resolved central serous chorioretinopathy.

We present a case of a patient with resolved central serous chorioretinopathy (CSC) in the left eye with persistent mild visual alterations 4 years after the resolution of the disease. Left eye exam revealed a best corrected visual acuity of 20/25 and a slight change of macular pigmentation. Optical coherence tomography revealed only minor irregularities in the topography of retinal pigment epithelium and Bruch's membrane. Adaptive optics (AO) optics demonstrated lower density, spacing, and changes in the photoreceptor mosaic pattern in the left eye than in the right eye, suggesting that CSC may cause damage to cones after clinical recovery. We conclude that AO can provide additional information to assist in the treatment and follow-up of patients with CSC or other macular pathologies.

Versatile functional roles of horizontal cells in the retinal circuit.

In the retinal circuit, environmental light signals are converted into electrical signals that can be decoded properly by the brain. At the first synapse of the visual system, information flow from photoreceptors to bipolar cells is modulated by horizontal cells (HCs), however, their functional contribution to retinal output and individual visual function is not fully understood. In the current study, we investigated functional roles for HCs in retinal ganglion cell (RGC) response properties and optokinetic responses by establishing a HC-depleted mouse line. We observed that HC depletion impairs the antagonistic center-surround receptive field formation of RGCs, supporting a previously reported HC function revealed by pharmacological approaches. In addition, we found that HC loss reduces both the ON and OFF response diversities of RGCs, impairs adjustment of the sensitivity to ambient light at the retinal output level, and alters spatial frequency tuning at an individual level. Taken together, our current study suggests multiple functional aspects of HCs crucial for visual processing.

Photoreceptor assessment using adaptive optics in resolved central serous chorioretinopathy / Avaliação dos fotorreceptores com adaptive optics na coriorretinopatia serosa central cicatrizada

ABSTRACT We present a case of a patient with resolved central serous chorioretinopathy (CSC) in the left eye with persistent mild visual alterations 4 years after the resolution of the disease. Left eye exam revealed a best corrected visual acuity of 20/25 and a slight change of macular pigmentation. Optical coherence tomography revealed only minor irregularities in the topography of retinal pigment epithelium and Bruch's membrane. Adaptive optics (AO) optics demonstrated lower density, spacing, and changes in the photoreceptor mosaic pattern in the left eye than in the right eye, suggesting that CSC may cause damage to cones after clinical recovery. We conclude that AO can provide additional information to assist in the treatment and follow-up of patients with CSC or other macular pathologies.

RESUMO Apresentamos o relato de caso de paciente com coriorretinopatia serosa central (CSC) cicatrizada em olho esquerdo e queixa de discreta alteração visual, mesmo após quatro anos da resolução do quadro. O exame do olho esquerdo apresenta melhor acuidade visual corrigida de 20/25 e discreta alteração de pigmentação macular. Tomografia de coerência óptica (OCT) apresentou apenas pequenas irregularidades em topografia de EPR e Bruch. Foi realizado exame com Adaptive Optics (AO), evidenciando valores inferiores de densidade, espaçamento e alterações no padrão de mosaico dos fotorreceptores em olho esquerdo quando comparado com olho direito, sugerindo que a CSC pode causar danos em cones, mesmo após uma recuperação considerada satisfatória. Concluímos que o AO é uma tecnologia que traz novas informações para auxiliar o tratamento e seguimento dos pacientes com CSC ou outras patologias maculares.

Postoperative Recovery of Light Sensitivity in Eyes with Rhegmatogenous Retinal Detachment.

PURPOSE: To investigate whether postoperative light sensitivity recovers completely to the level prior to the development of rhegmatogenous retinal detachment (RRD) after successful surgery. METHODS: We retrospectively studied 44 eyes of 44 patients with RRD who were successfully operated and who underwent Humphrey central 30-2 perimetry postoperatively. The averaged total deviation in Humphrey perimetry in the reattached retina was compared with that of the horizontal or vertical counterpart in the preoperatively non-detached retina. RESULTS: The averaged total deviation in the reattached retina was significantly lower than in its counterpart (p < 0.0001). The averaged residual loss of light sensitivity did not correlate with postoperative visual acuity (p = 0.8047) or with its change (p = 0.1242). CONCLUSIONS: Light sensitivity in the detached retina in eyes with RRD does not completely recover after successful surgery.

The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones.

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones.

Regeneration of Photoreceptor Outer Segments After Scleral Buckling Surgery for Rhegmatogenous Retinal Detachment.

PURPOSE: To investigate the regeneration of the cone outer segments in eyes after surgery for fovea-off rhegmatogenous retinal detachment with an adaptive optics (AO) fundus camera and to correlate these findings with the findings of optical coherence tomography (OCT). DESIGN: Retrospective, observational case series. METHODS: Medical charts of 21 eyes of 21 patients who had undergone surgery for fovea-off rhegmatogenous retinal detachment were retrospectively studied. Cone mosaic images were obtained with an AO fundus camera. Cone packing density at 2 degrees from the fovea within the previously detached area was measured 6 and 12 months after surgery. Retinal thicknesses between the interdigitation zone and the retinal pigment epithelium (IZ-RPE) and between the ellipsoid zone and the retinal pigment epithelium (EZ-RPE) were measured in OCT images. RESULTS: Cone density 12 months after surgery was significantly increased from that at 6 months (P = .001), but was still significantly lower than that of normal fellow eyes (P < .001). IZ-RPE and EZ-RPE thickness significantly increased from 6 to 12 months (P = .045, P = .033, respectively), and these values were not significantly different from those of normal fellow eyes. Multivariate analysis showed that cone density at 12 months was significantly associated with IZ-RPE thickness (P = .002), and increases in cone packing density were significantly associated with increases in IZ-RPE thickness (P = .001). CONCLUSIONS: Recovery of cone packing density measured by AO was associated with structural recovery of the outer retina observed in OCT, suggesting regeneration of the photoreceptor outer segment after surgery.

The contribution of inner and outer retinal photoreceptors to infra-slow oscillations in the rat olivary pretectal nucleus.

A subpopulation of olivary pretectal nucleus (OPN) neurons discharges action potentials in an oscillatory manner, with a period of approximately two minutes. This 'infra-slow' oscillatory activity depends on synaptic excitation originating in the retina. Signals from rod-cone photoreceptors reach the OPN via the axons of either classic retinal ganglion cells or intrinsically photosensitive retinal ganglion cells (ipRGCs), which use melanopsin for photon capturing. Although both cell types convey light information, their physiological functions differ considerably. The aim of the present study was to disentangle how rod-cone and melanopsin photoresponses contribute to generation of oscillatory activity. Pharmacological manipulations of specific phototransduction cascades were used whilst recording extracellular single-unit activity in the OPN of anaesthetized rats. The results show that under photopic conditions (bright light), ipRGCs play a major role in driving infra-slow oscillations, as blocking melanopsin phototransmission abolishes or transiently disturbs oscillatory firing of the OPN neurons. On the other hand, blocking rod-cone phototransmission does not change firing patterns in photopic conditions. However, under mesopic conditions (moderate light), when melanopsin phototransmission is absent, blocking rod-cone signalling causes disturbances or even the disappearance of oscillations implying that classic photoreceptors are of greater importance under moderate light. Evidence is provided that all photoreceptors are required for the generation of oscillations in the OPN, although their roles in driving the rhythm are determined by the lighting conditions, consistent with their relative sensitivities. The results further suggest that maintained retinal activity is crucial to observe infra-slow oscillatory activity in the OPN.