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Background: Mild traumatic brain injury (mTBI)is the most common form of traumatic brain injury accounting for 70-80% of all brain injuries annually. There is increasing evidence that long lasting morphological and functional consequence can be present in visual system following mTBI. Among all the visual manifestation, awareness of Visual field defects is important because it may compromise the social, personal or professional life of any individual. Retinal structural changes such as thinning of Retinal nerve fiber layer (RFNL)captured using optical coherence tomography have emerged as a possible biomarker in many neurological diseases however very little is known in cases with mTBI OBJECTIVE: (I) To demonstrate the structural changes/morphological changes in retina if any following mTBI. (II) Whether the structural changes in retina have any association with the development of Visual field deficits leading to Visual function impairment following mTBI (III) Clinical relevance of structural changes in retina as a possible biomarker for visual function impairment due to visual field deficits., Materials and Methods: Our study included 60 patients with mTBI who fulfilled the inclusion criteria. All patients underwent a detailed ophthalmic evaluation with special focus on temporal recording of Retinal nerve layer thickness using SD- Optical Coherence Tomography and Visual field (Visual field Index) by Humphrey Automated Field Analyser., Results: 30% of eyes had significant thinning of RFNL (> 30% of the base line thickness) at 6 months following mTBI. Visual function impairment due to visual field deficits (VFI < 80%) at 6 months was seen in 40% of the eyes. The structural changes and visual function impairment peaked at 6 months' post injury. A strong Association was noted between RFNL thinning and manifestation of Visual field deficits (VFI < 80%) leading to visual function impairment (P < 0.001). The Correlation Co-efficient between thinning of RFNL and Visual field deficits had a positive correlation(p < 0.001)., Conclusion: This novel study has demonstrated that visual functional impairment due to Visual field deficits is a real possibility following mTBI. Monitoring of retinal parameter such as thinning of Retinal nerve fiber layer, using Optical coherence tomography, can be a biomarker for early detection or development of visual field defects in mTBI., (Copyright © 2021. Published by Elsevier B.V.)

It is well established that the adult mammalian pineal body (PB), with the exception of rodents, contains nerve cell bodies. Based on our previous results we have proposed that there is a pinealo-to-retinal neuronal connection in adult hamsters and in prebubertal rats. By the time the animals reached puberty, labeled cells in the PB were not observed in rats. In the present experiment, we provide light and electron microscopic immunohistochemical evidence that the labeled cells in the PB of prepubertal rats are neurons. Pinealocytes cannot transport neurotropic viruses. Virus labeled cells do not show S-antigen immunoreactivity typical for pinealocytes of six-day-old rats. Electron microscopic investigation confirmed the neuronal nature of virus labeled cells. These neurons, similarly to that of hamsters, also establish pinealo-to-retinal connections in prepubertal rats., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Retinal ischemia, one of the most common cause of visual loss, is associated with blood flow inadequacy and subsequent tissue injury. In this setting, some treatments that can counteract glutamate increase, arouse interest in ischemic pathogenesis. Ketamine, a potent N-methyl-d-aspartate (NMDA) receptor antagonist, provides a neuroprotective pathway via decreasing the excitotoxicity triggered by excess glutamatergic. Thus, the goal of this study was to evaluate the safety of intravitreal use of ketamine and their potential protective effects on retinal cells in retinal ischemia/reperfusion model. Initially, ketamine toxicity was evaluated by cytotoxicity assay and Hen's egg chorioallantoic membrane (HET-CAM) method. Afterward, some ketamine concentrations were tested in rat's eyes to verify the safety of the intravitreal use. To investigate the neuroprotective effect on retinal, a single intravitreal injection of ketamine in concentrations of 0.059 mmol.L -1 and 0.118 mmol.L -1 was performed one day before the retinal injury by ischemia/reperfusion model. After 7 and 15 days, the retina activity was evaluated by electroretinogram (ERG) records and, lastly, by morphological analyzes. Cytotoxicity assay reveals that the maximum ketamine concentration that could reach retinal pigmented epithelium cells is 0.353 mmol.L -1 . HET-CAM assay showed that concentrations above 0.237 mmol.L -1 are irritants to the eye. Thus, Ketamine in concentrations of 0.0237 mmol.L -1 , 0.118 mmol.L-1, and 0.059 mmol.L-1 were selected for in vivo toxicity test. ERG records reveal a tendency of b-wave amplitude to decrease as the luminous intensity increased, in the group receiving ketamine at 0.237 mmol.L -1 . Therefore, ketamine in concentrations at 0.059 mmol.L -1 and 0.118 mmol.L-1 were chosen for the following tests. In the ischemia retinal degeneration model, pretreatment with ketamine was capable to promote a recovery of retinal electrophysiological function minimizing the ischemic effects. In histological analysis, the groups that received intravitreal ketamine showed a number of retinal cells significantly higher than the vehicle group. In TUNEL assay a reduction on TUNEL-positive cells was observed in all the layers for both concentrations which allow to affirm that ketamine contributes to reducing cell death in the retina. Transmission electron microscopy (TEM) reaffirms this finding. Ketamine intravitreal pretreatment showed reduced ultrastructural changes. Our findings demonstrate that ketamine is safe for intravitreal use in doses up to 0.118 mmol.L -1 . They seem to be particularly efficient to protect the retina from ischemic injury., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background and Objectives : the aim of the study was to evaluate the repeatability and reproducibility of optical microangiography (OMAG)-based optical coherence tomography angiography (OCTA) in the optic nerve head (ONH) and radial peripapillary capillary (RPC) perfusion assessment of healthy eyes. Materials and Methods : in this observational study, a total of 40 healthy subjects underwent ONH evaluation, using an OMAG-based OCTA system at baseline (T 0 ), after 30 min (T 1 ), and after 7 days (T 2 ). The main outcome measures were the vessel density (VD) and flux index (FI) of the RPCs, as well as peri-papillary retinal nerve fibre layer (pRNFL) thickness. The analysis was performed by two observers independently. The coefficient of repeatability (CR), within the subject coefficient of variation (CVw) and intrasession correlation coefficient (ICC), to evaluate intrasession repeatability of measurements was calculated for each observer. Results : the high intrasession and intersession repeatability and reproducibility were assessed in the two observers for all three outcome measures. Of note, the CRs for the first and the second observer were 0.011 (95% confidence interval (CI) 0.009-0.014) and 0.016 (95% CI 0.013-0.020) for FI, 0.016 (95% CI 0.013-0.021) and 0.017 (95% CI 0.014-0.021) for VD, and 2.400 (95% CI 1.948-3.092) and 3.732 (95% CI 3.064-4.775) for pRNFL thickness, respectively. The agreement between them was excellent for pRNFL assessment and very good for FI and VD. Conclusion : OCTA has a great potential in the accurate assessment of ONH and peri-papillary microcirculation. It allows for repeated and reproducible measurements without multiple scans-related bias, thus guaranteeing an independent operator analysis with good reproducibility and repeatability.

Aims: To investigate the association between progressive macular ganglion cell/inner plexiform layer (mGCIPL) thinning and change of optical coherence tomography angiography (OCTA)-derived microvascular parameters in early-stage diabetic retinopathy (DR)., Methods: A retrospective cohort study involved 40 eyes presenting with no DR or mild non-proliferative DR at baseline, and 30 healthy controls were included. All participants underwent spectral-domain OCT and OCTA at baseline and at 6, 12, 18, and 24 months. Change of mGCIPL thickness and OCTA metrics including foveal avascular zone (FAZ) area and FAZ circularity, vessel density (VD), and perfusion index (PI) was measured. Correlations between mGCIPL thickness and OCTA metrics were explored using regression models., Results: Average progressive mGCIPL loss was 0.45 µm per year. Three microvascular parameters were significantly impaired at 24 months compared to baseline (FAZ area: 0.34-0.36 mm 2 , VD: 18.9-18.5/mm, PI: 0.35-0.34). A strong positive correlation was found between loss of mGCIPL and VD from baseline to 24 months (r = 0.817, p < 0.001). Multivariable regression analysis showed that thinner baseline mGCIPL and greater loss of mGCIPL thickness (B = 0.658, p < 0.001) were significantly associated with change of VD., Conclusions: In the early stage of DR, progressive structural retinal neurodegeneration and parafoveal microvascular change seem to be highly linked. Advanced mGCIPL thinning might precede microvascular impairment in early DR.

Purpose: The purpose of this study was to compare the peripapillary retinal nerve fibre layer (RNFL) between patients with genetic generalized epilepsy (GGE) and healthy controls., Methods: This prospective observational study was conducted on adults aged 18-60 years. The study group comprised 26 consecutive patients who met the inclusion criteria and 26 healthy age- and sex-matched healthy adults. Peripapillary RNFL thickness was measured by spectral domain optical coherence tomography., Results: The average peripapillary RNFL thickness was significantly thinner for GGE patients (98.61 μm) than for healthy controls (104.77 μm) (p = 0.016). Similar results were obtained for the left eye. The peripapillary RFNL thickness of all quadrants was lower for GGE patients than for healthy controls, but it was significant only in the superior (p = 0.009) and inferior (p = 0.024) quadrants for both eyes., Conclusions: Our results suggest that the peripapillary RNFL is significantly thinner in GGE patients than in healthy participants. We concluded that this microstructural feature might be an intrinsic feature of GGE., (Copyright © 2019 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones. SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly., (Copyright © 2019 the authors.)

Objective: To evaluate neural and vascular retinal morphology of children with isolated growth hormone deficiency (GHD) and to determine any retinal changes due to GH treatment., Methods: Twenty-eight children with isolated GHD and 53 age-, gender- and body mass index-matched healthy volunteers were enrolled in this prospective study. The retinal nerve fibre layer (RNFL) and macular thickness (MT) were measured, as well as intraocular pressure (IOP). The number of retinal vascular branching points were calculated. Effect of GH treatment on the retina and IOP was evaluated after one year of treatment. Measurements were also made in the control group at baseline and following the initial examination. Pre- and post-treatment changes were compared. The findings were also compared with those of the controls. The correlation between ocular dimensions and insulin-like growth factor-I (IGF-1) levels were also analysed., Results: The number of branching points was significantly lower in GHD patients as compared with control subjects (15.11±2.67 and 19.70±3.37, respectively, p=0.05 for all comparisons). No statistically significant differences were found in mean RNFL, MT and IOP values between GHD patients and control subjects. GH treatment did not create any significant changes in the retinal vascularization or other retinal neural parameters and IOP either within the patient group or when compared with the control group. No correlations were observed between ocular dimensions and IGF-1 levels., Conclusion: Our findings suggest that isolated GHD may lead to decreased retinal vascularization. However, retinal neural growth and differentiation were not affected by GHD. These findings may be related to the fetal development process of pituitary somatotropic cells and the retina. Additionally, GH treatment did not cause any changes in retinal neural and vascular tissues.

The mouse retina has a layered structure that is composed of five classes of neurons supported by Müller glial and pigment epithelial cells. Recent studies have made progress in the classification of bipolar and ganglion cells, and also in the wiring of rod-driven signaling, color coding, and directional selectivity. Molecular biological techniques, such as genetic manipulation, transcriptomics, and fluorescence imaging, have contributed a lot to these advancements. The mouse retina has consistently been an important experimental system for both basic and clinical neurosciences.

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.

Polyamines are tightly regulated polycations that are essential for life. Loss-of-function mutations in spermine synthase (SMS), a polyamine biosynthesis enzyme, cause Snyder-Robinson syndrome (SRS), an X-linked intellectual disability syndrome; however, little is known about the neuropathogenesis of the disease. Here we show that loss of dSms in Drosophila recapitulates the pathological polyamine imbalance of SRS and causes survival defects and synaptic degeneration. SMS deficiency leads to excessive spermidine catabolism, which generates toxic metabolites that cause lysosomal defects and oxidative stress. Consequently, autophagy-lysosome flux and mitochondrial function are compromised in the Drosophila nervous system and SRS patient cells. Importantly, oxidative stress caused by loss of SMS is suppressed by genetically or pharmacologically enhanced antioxidant activity. Our findings uncover some of the mechanisms underlying the pathological consequences of abnormal polyamine metabolism in the nervous system and may provide potential therapeutic targets for treating SRS and other polyamine-associated neurological disorders.

Aims: To analyze retinal vascular plexuses and choriocapillaris by optical coherence tomography angiography (OCT-A) and retinal nerve fiber layer and ganglion cell layer (GCL) by structural optical coherence tomography (OCT) in patients with type 1 diabetes mellitus (T1DM) without diabetic retinopathy (DR)., Methods: A total of 25 eyes of 25 consecutive T1DM patients without signs of DR were prospectively recruited and compared to 25 healthy subjects (control eyes). All patients underwent OCT-A (CIRRUS HD-OCT model 5000, Carl Zeiss Meditec, Dublin, CA) and structural OCT. Qualitative and quantitative analyses with vessel density were performed on OCT-A images in the superficial capillary plexus (SCP), deep capillary plexus (DCP) and choriocapillaris for all patients., Results: By means of OCT-A, a rarefaction of the perifoveal capillary network in SCP was detected in 7 out of 25 eyes. No significant difference was found in FAZ area of both SCP and DCP comparing diabetic and control groups. By analyzing the DCP, diabetic eyes revealed a significant decreased vessel density compared to control eyes [0.464 ± 0.016 and 0.477 ± 0.014, respectively (p = 0.005)]. Instead, no significant difference was found in the vessel density of all-retina plexus, SCP and choriocapillaris. By RFNL and GCL thickness analysis, no significant differences were disclosed between diabetics and healthy subjects., Conclusions: We demonstrated the ability of OCT-A to disclose early vascular alterations in patients with T1DM diagnosed as without any signs of DR on the basis of fundus biomicroscopy. Our results also suggest that microvascular changes could precede detectable damage of diabetic neuroretinopathy.

Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell-specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear. Here, we have created a tissue-specific DNA methyltransferase (Dnmt) triple mutant mouse in an effort to characterize the impact of DNA methylation on retinal development and homeostasis. An Rx-Cre transgene was used to drive targeted mutation of all three murine Dnmt genes in the mouse retina encoding major DNA methylation enzymes DNMT1, DNMT3A and DNMT3B. The triple mutant mice represent a hypomorph model since Dnmt1 catalytic activity was still present and excision of Dnmt3a and Dnmt3b had only about 90% efficiency. Mutation of all three Dnmts resulted in global genomic hypomethylation and dramatic reorganization of the photoreceptor and synaptic layers within retina. Transcriptome and proteomic analyses demonstrated enrichment of dysregulated phototransduction and synaptic genes. The 5 mC signal in triple mutant retina was confined to the central heterochromatin but reduced in the peripheral heterochromatin region of photoreceptor nuclei. In addition, we found a reduction of the 5 mC signal in ganglion cell nuclei. Collectively, this data suggests cooperation of all three Dnmts in the formation and homeostasis of photoreceptors and other retinal neurons within the mammalian retina, and highlight the relevance of epigenetic regulation to sensory retinal disorders and vision loss., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Identification of the key components in the physical and chemical milieu directing donor cells into a desired phenotype is a requirement in the investigation of bioscaffolds for the advancement of cell-based therapies for retinal neurodegeneration. We explore the effect of electrospun poly-ε-caprolactone (PCL) fiber scaffold topography and functionalization and culture medium, on the behavior of mouse retinal cells. Dissociated mouse retinal post-natal cells were seeded on random or aligned oriented fibers, with or without laminin coating and cultured with either basic or neurotrophins enriched medium for 7days. Addition of laminin in combination with neurotrophins clearly promoted cell- morphology, fate, and neurite extension. Nanotopography per se significantly affected cell morphology, with mainly bipolar profiles on aligned fibers and more multipolar profiles on random fibers. Laminin induced a remarkable 90° switch of neurite orientation. Herewith, we demonstrate that the chemical cue is stronger than the physical cue for the orientation of retinal neurites and describe the requirement of both neurotrophins and extracellular matrix proteins for extended neurite outgrowth and formation of complex retinal neuronal networks. Therefore, tailor-made PCL fiber mats, which can be physically and chemically modified, indeed influence cell behavior and hence motivate further retinal restorative studies using this system., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: We examined the relationship between retinal nerve fiber layer (RNFL) thickness and cognitive function in a population of older British adults., Methods: Participants of the European Prospective Investigation of Cancer (EPIC) Norfolk cohort study underwent ophthalmic and cognitive assessment. Measurements of RNFL thickness were made using the Heidelberg Retina Tomograph (HRT). Cognitive testing included a short form of the Mini-Mental State Examination (SF-MMSE), an animal naming task, a letter cancellation task, the Hopkins Verbal Learning Test (HVLT), the National Adult Reading Test (NART), and the Paired Associates Learning Test. Multivariable linear regression models were used to assess associations of RNFL thickness with cognitive test scores, adjusted for age, sex, education level, social class, visual acuity, axial length, and history of cataract surgery., Results: Data were available from 5563 participants with a mean age of 67 years. A thicker HRT-derived RNFL thickness was associated with better scores for the SF-MMSE (0.06; 95% confidence interval [CI], [0.02, 0.10], P = 0.005), HVLT (0.16, 95% CI [0.03, 0.29]; P = 0.014), and NART (-0.24, 95% CI [-0.46, -0.02], P = 0.035). The associations of RNFL thickness with SF-MMSE and HVLT remained significant following further adjustment for NART., Conclusions: We found a significant association between HRT-derived RNFL thickness and scores from cognitive tests assessing global function, recognition, learning, episodic memory, and premorbid intelligence. However, the associations were weak and not currently of predictive value. Further research is required to confirm and clarify the nature of these associations, and identify biological mechanisms.

The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations. We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina. Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well. A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons.

Neuromyelitis optica (NMO) is an inflammatory autoimmune disease of the central nervous system that preferentially targets the optic nerves and spinal cord. The clinical presentation may suggest multiple sclerosis (MS), but a highly specific serum autoantibody against the astrocytic water channel aquaporin-4 present in up to 80% of NMO patients enables distinction from MS. Optic neuritis may occur in either condition resulting in neuro-anatomical retinal changes. Optical coherence tomography (OCT) has become a useful tool for analyzing retinal damage both in MS and NMO. Numerous studies showed that optic neuritis in NMO typically results in more severe retinal nerve fiber layer (RNFL) and ganglion cell layer thinning and more frequent development of microcystic macular edema than in MS. Furthermore, while patients' RNFL thinning also occurs in the absence of optic neuritis in MS, subclinical damage seems to be rare in NMO. Thus, OCT might be useful in differentiating NMO from MS and serve as an outcome parameter in clinical studies., (© The Author(s), 2015.)

The 36-week ATON study compared the efficacy and safety of atacicept with matching placebo in 34 patients with unilateral optic neuritis as a clinically isolated syndrome. Atacicept (150mg) was administered twice weekly for 4weeks (loading period), then once weekly for 32weeks. The ATON study was terminated prematurely by the sponsor when an independent Data and Safety Monitoring Board review observed increased multiple sclerosis (MS)-related disease activity in the atacicept arms of the concurrent ATAcicept in MS (ATAMS) study. Analysis of the prematurely terminated ATON study showed that the mean (standard deviation) change from baseline in retinal nerve fiber layer thickness at last observed value in the affected eye was -8.6 (10.1) μm in patients treated with atacicept (n=15) compared with -17.3 (15.2) μm in patients treated with placebo (n=16). In the atacicept treatment group, a higher proportion of patients converted to clinically definite MS during the double-blind period compared with placebo (35.3% [6/17] vs 17.6% [3/17]). Treatment-emergent adverse events were similar across both treatment groups in the double-blind period. A dichotomy emerged with more atacicept-treated patients converting to relapsing-remitting MS compared with placebo-treated patients, despite the same patients experiencing less axonal loss after an optic neuritis event., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N=9), dementia with Lewy bodies (DLB; N=3), Alzheimer's disease (N=3), progressive supranuclear palsy (N=2) as well as elderly normal control subjects (N=4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Mouse retinal degeneration models have been investigated for many years in the hope of understanding the mechanism of photoreceptor cell death. N -methyl- N -nitrosourea (MNU) has been previously shown to induce outer retinal degeneration in mice. After MNU was intraperitoneally injected in C57/BL mice, we observed a gradual decrease in the outer nuclear layer (ONL) thickness associated with photoreceptor outer segment loss, bipolar cell dendritic retraction and reactive gliosis. Reactive gliosis was confirmed by increased GFAP protein levels. More serious damage to the central retina as opposed to the peripheral retina was found in the MNU-induced retinal degeneration model. Retinal ganglion cells (RGC) appear to be spared for at least two months after MNU treatment. Following retinal vessel labelling, we observed vascular complexes in the distal vessels, indicating retinal vessel damage. In the remnant retinal photoreceptor of the MNU-treated mouse, concentrated colouring nuclei were detected by electron microscopy, together with the loss of mitochondria and displaced remnant synaptic ribbons in the photoreceptor. We also observed decreased mitochondrial protein levels and increased amounts of nitrosylation/nitration in the photoreceptors. The mechanism of MNU-induced apoptosis may result from oxidative stress or the loss of retinal blood supply. MNU-induced mouse retinal degeneration in the outer retina is a useful animal model for photoreceptor degeneration diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Light-emitting diodes (LEDs) deliver higher levels of blue light to the retina than do conventional domestic light sources. Chronic exposure to high-intensity light (2,000-10,000 lux) has previously been found to result in light-induced retinal injury, but chronic exposure to relatively low-intensity (750 lux) light has not been previously assessed with LEDs in a rodent model., Objective: We examined LED-induced retinal neuronal cell damage in the Sprague-Dawley rat using functional, histological, and biochemical measurements., Methods: We used blue LEDs (460 nm) and full-spectrum white LEDs, coupled with matching compact fluorescent lights, for exposures. Pathological examinations included electroretinogram, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and transmission electron microscopy (TEM). We also measured free radical production in the retina to determine the oxidative stress level., Results: H&E staining and TEM revealed apoptosis and necrosis of photoreceptors, which indicated blue-light induced photochemical injury of the retina. Free radical production in the retina was increased in LED-exposed groups. IHC staining demonstrated that oxidative stress was associated with retinal injury. Although we found serious retinal light injury in LED groups, the compact fluorescent lamp (CFL) groups showed moderate to mild injury., Conclusion: Our results raise questions about adverse effects on the retina from chronic exposure to LED light compared with other light sources that have less blue light. Thus, we suggest a precautionary approach with regard to the use of blue-rich "white" LEDs for general lighting., Citation: Shang YM, Wang GS, Sliney D, Yang CH, Lee LL. 2014. White light-emitting diodes (LEDs) at domestic lighting levels and retinal injury in a rat model. Environ Health Perspect 122:269-276; http://dx.doi.org/10.1289/ehp.1307294.

Purpose: To investigate the potential protective effects of total flavonoids from Flos Puerariae (TFF) on retinal neural cells in diabetic mice., Methods: C57BL/6J mice were intraperitoneally injected with streptozotocin to generate type I diabetes in a murine model, as indicated by blood glucose levels ≥11.1 mmol/l. TFF was administered intragastrically at a dose of 50, 100, or 200 mg/kg/day. After 10 weeks of administration, the mice were euthanized, and the eyes were dissected. Retinal histology was examined, and the thickness of the retina was measured. Ultrastructural changes in the retinal ganglion cells and capillary basement membrane were observed with electron microscopy. Apoptosis of retinal neural cells was determined with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay. Bax and Bcl-2 expression in the retinal tissues was determined with immunohistochemical staining and western blotting., Results: Compared with the diabetic mice, the blood glucose level decreased (p<0.01) and the bodyweight increased (p<0.05) in the 100 and 200 mg/kg TFF-treated groups. The thickness of the retina significantly increased (p<0.01), and the retinal capillary basement membrane (BM) thickness was reduced in the 100 and 200 mg/kg TFF-treated diabetic mice (DM). The 100 and 200 mg/kg TFF treatments also attenuated the diabetes-induced apoptosis of retinal neural cells. Consistent with these effects, TFF treatment decreased the Bax expression level and, concurrently, increased the ratio of Bcl-2 to Bax., Conclusions: TFF attenuated diabetes-induced apoptosis in retinal neurons by inhibiting Bax expression and increasing the ratio of Bcl-2 to Bax, which suggests that TFF might prevent retinal neuronal damage in diabetes mellitus.

Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute to the information processing in the retinal circuits. However, many of previous electrophysiological and immunocytochemical studies employed slice preparations cut by vertical direction in which most of these cells and their neurites were severely damaged and removed. This might lead to the underestimation of active and passive conductance in horizontally expanding neurites, and also missing of morphological information of horizontal structures. Here, we describe an alternative slicing method of horizontally cut preparation of the retina. The slice is made horizontally at the inner layer of the retina using a vibratome slicer after the retina is embedded in the low-temperature melting agarose gel. This horizontal slice preparation enables us to directly access cells in the inner retina by patch-clamp recording, calcium imaging, single RT-PCR, and immunocytochemistry. The method described here would offer an alternative strategy for studying the functions of neurons and neural circuits in the retina.

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide, acting as a neuromodulator and neuroprotective peptide in the CNS after injuries. We have previously described that pituitary adenylate cyclase-activating polypeptide (PACAP), another member of the same peptide family, is retinoprotective in ischemic lesions. The aim of this study was to investigate the protective potential of VIP in bilateral common carotid artery occlusion (BCCAO)-induced ischemic retinal lesion. Two-month-old rats were subjected to BCCAO and treated with intravitreal VIP injection. Their retinas were processed for histology after 2 weeks of survival. We measured the number of the cells/100 μm of the ganglion cell layer and the thickness of each layer such as the outer nuclear, outer plexiform, inner nuclear, and inner plexiform layers as well as that of the whole retina. We found that treatment with 1,000 pmol VIP, but not 100 pmol VIP, had significant protective effects in BCCAO-injured retina, as shown by the morphometric analysis. Comparing the neuroprotective effects of VIP and PACAP in BCCAO-operated retinas, PACAP was more effective, already protective at 100-pmol doses. Similar to other studies, we found that VIP must be given at least in 10 times more concentration than PACAP to achieve a similar degree of neuroprotection in the retina.

The widely used botulinum neurotoxin A (BoNT/A) blocks neurotransmission via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Recent evidence demonstrating long-distance propagation of SNAP-25 proteolysis has challenged the idea that BoNT/A remains localized to the injection site. However, the extent to which distant neuronal networks are impacted by BoNT/A retrograde trafficking remains unknown. Importantly, no studies have addressed whether SNAP-25 cleavage translates into structural and functional changes in distant intoxicated synapses. Here we show that the BoNT/A injections into the adult rat optic tectum result in SNAP-25 cleavage in retinal neurons two synapses away from the injection site, such as rod bipolar cells and photoreceptors. Retinal endings displaying cleaved SNAP-25 were enlarged and contained an abnormally high number of synaptic vesicles, indicating impaired exocytosis. Tectal injection of BoNT/A in rat pups resulted in appearance of truncated-SNAP-25 in cholinergic amacrine cells. Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity, demonstrating impairments in neurotransmission. These data provide the first evidence for functional effects of the retrograde trafficking of BoNT/A, and open the possibility of using BoNT/A fragments as drug delivery vehicles targeting the central nervous system., (© 2012 John Wiley & Sons A/S.)

Objective: To assess retinal nerve fiber (RNFL) thickness change in glaucoma patients after glaucoma surgery., Material and Method: A prospective and cohort study of 19 patients who underwent trabeculectomy with mitomicin C or combined procedures or glaucoma drainage device surgery in Phramongkutklao Hospital were included. All subjects had obtained informed consents. Preoperative and 3 month-postoperative IOP and RNFL thickness were determined. Areas of peripapilllary RNFL thickness were evaluated by Stratus OCT scanning. The relationship between IOP reduction and RNFL thickness change after glaucoma surgery was analyzed., Results: Postoperative RNFL thickness analysis found no significant increase in inferior, superior, nasal and temporal quadrants. The average of preoperative RNFL thickness was 59.58 +/- 17.59 microns, whereas the average of postoperative RNFL thickness was 57.19 +/- 14.97 microns (p = 0.426). Postoperative IOP was decreased significantly from 29.26 +/- 8.58 mmHg to 12.11 +/- 4.52 mmHq (p < 0.001). The mean of postoperative RNFL thickness was not significantly correlated with IOP reduction (r = 0.306, p = 0.250)., Conclusion: Post glaucoma surgery, RNFL thickness was not significantly increased although the IOP had significantly reduced. No correlation between IOP reduction and RNFL thickness was found.

Purpose: To conduct high-resolution imaging of the retinal nerve fiber layer (RNFL) in normal eyes using adaptive optics scanning laser ophthalmoscopy (AO-SLO)., Methods: AO-SLO images were obtained in 20 normal eyes at multiple locations in the posterior polar area and a circular path with a 3-4-mm diameter around the optic disc. For each eye, images focused on the RNFL were recorded and a montage of AO-SLO images was created., Results: AO-SLO images for all eyes showed many hyperreflective bundles in the RNFL. Hyperreflective bundles above or below the fovea were seen in an arch from the temporal periphery on either side of a horizontal dividing line to the optic disc. The dark lines among the hyperreflective bundles were narrower around the optic disc compared with those in the temporal raphe. The hyperreflective bundles corresponded with the direction of the striations on SLO red-free images. The resolution and contrast of the bundles were much higher in AO-SLO images than in red-free fundus photography or SLO red-free images. The mean hyperreflective bundle width around the optic disc had a double-humped shape; the bundles at the temporal and nasal sides of the optic disc were narrower than those above and below the optic disc (P<0.001). RNFL thickness obtained by optical coherence tomography correlated with the hyperreflective bundle widths on AO-SLO (P<0.001), Conclusions: AO-SLO revealed hyperreflective bundles and dark lines in the RNFL, believed to be retinal nerve fiber bundles and Müller cell septa. The widths of the nerve fiber bundles appear to be proportional to the RNFL thickness at equivalent distances from the optic disc.

Objectives: Mild cognitive impairment (MCI) may represent a transition to early Alzheimer's disease (AD). The retinal nerve fiber layer (RNFL) is composed of axons originating in retinal ganglion cells that eventually form the optic nerves. Previous studies have shown that degenerative changes occur in optic nerve fibers and manifested as thinning of RNFL in patients with AD. The objective of this study was to assess the relationship between MCI, AD and loss of RNFL., Patients and Methods: In this study, patients fulfilling diagnostic criteria for MCI (n=24), AD (n=30) and cognitively normal age-matched controls (n=24) have undergone neuro-ophthalmologic and optical coherence tomography (OCT) examinations to measure RNFL thickness., Results: There was a significant decrease in RNFL thickness in both study groups (AD and MCI) compared to the control group, particularly in the inferior quadrants of the optic nerve head, while the superior quadrants were significantly thinner only in AD. Although AD patients may have more severe changes than MCI cases, the differences were statistically nonsignificant. Furthermore among AD patients, there was no relation to the severity of the dementia., Conclusions: Our data confirm the retinal involvement in AD, as reflected by loss of axons in the optic nerves., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the neural retina, glial cells control the ionic concentrations in part by mediation of transmembrane water fluxes through aquaporin (AQP) water channels. The expression and immunolocalization of two water channels, AQP1 and AQP4, in the rat retina during experimental high salt loading were investigated in this study. Six-week-old Wistar rats were allowed free access to rat chow with 8% NaCl concentration. Of these rats, 6 were killed after 2, 6, 10 and 20 weeks. Twelve-week-old and 26-week-old Wistar rats with a normal diet (0.5% NaCl concentration) were used as controls. Retinal tissues were collected. Ultrathin sections stained with uranyl acetate and lead citrate were photographed using a transmission electron microscope (TEM). Retinal whole mounts and cryosections were immunostained with AQP1 and AQP4 antibodies to detect the immunolocalization changes by confocal microscopy. The AQP1 and AQP4 contents were evaluated by western blot analysis. In control tissues, no intracellular edema and mitochondria swelling were observed by TEM. The immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina. In the retinas of high salt loading animals, obvious intracellular edema was observed by TEM in retinal ganglion cell (RGC) and mitochondria swelling was observed in glial cells. Strong expression of AQP1 was found in glial cells located in the innermost retinal layers, mainly in astrocytes. The superficial retinal vessels were surrounded by AQP4 in control retinas, but by both AQP4 and AQP1 in retina of high salt loading animals. A similar alteration in the localization of AQP1 has been described in the rat retina after transient ischemia and diabetes. Western blot results supported the conclusion that the AQP1 expression increased during high salt diet. Our findings indicate that high salt loading may induce neural retina edema, and that altered glial cell-mediated water transport via AQP channels in the retina may be one of the reasons for intracellular edema in the neural retina.

Circuitry mapping of metazoan neural systems is difficult because canonical neural regions (regions containing one or more copies of all components) are large, regional borders are uncertain, neuronal diversity is high, and potential network topologies so numerous that only anatomical ground truth can resolve them. Complete mapping of a specific network requires synaptic resolution, canonical region coverage, and robust neuronal classification. Though transmission electron microscopy (TEM) remains the optimal tool for network mapping, the process of building large serial section TEM (ssTEM) image volumes is rendered difficult by the need to precisely mosaic distorted image tiles and register distorted mosaics. Moreover, most molecular neuronal class markers are poorly compatible with optimal TEM imaging. Our objective was to build a complete framework for ultrastructural circuitry mapping. This framework combines strong TEM-compliant small molecule profiling with automated image tile mosaicking, automated slice-to-slice image registration, and gigabyte-scale image browsing for volume annotation. Specifically we show how ultrathin molecular profiling datasets and their resultant classification maps can be embedded into ssTEM datasets and how scripted acquisition tools (SerialEM), mosaicking and registration (ir-tools), and large slice viewers (MosaicBuilder, Viking) can be used to manage terabyte-scale volumes. These methods enable large-scale connectivity analyses of new and legacy data. In well-posed tasks (e.g., complete network mapping in retina), terabyte-scale image volumes that previously would require decades of assembly can now be completed in months. Perhaps more importantly, the fusion of molecular profiling, image acquisition by SerialEM, ir-tools volume assembly, and data viewers/annotators also allow ssTEM to be used as a prospective tool for discovery in nonneural systems and a practical screening methodology for neurogenetics. Finally, this framework provides a mechanism for parallelization of ssTEM imaging, volume assembly, and data analysis across an international user base, enhancing the productivity of a large cohort of electron microscopists., Competing Interests: Competing interests. Robert E. Marc is a principal of Signature Immunologics. All other authors declare no other competing interests.

The adult mammalian retina has for long been considered to lack a neurogenerative capacity. However, retinal stem/progenitor cells, which can originate retinal neurons in vitro, have been recently reported in the ciliary body of adult mammals. Here we explored the possibility of retinal neurogenesis occurring in vivo in adult monkeys and humans. We found the presence of cells expressing molecular markers of neural and retinal progenitors in the nonlaminated retinal margin and ciliary body pars plana of mature primates. By means of immunohistochemistry and electron microscopy we also observed photoreceptors and other retinal cell types in different stages of morphological differentiation along the peripheral retinal margin. These findings allow us to extend to primates the idea of neurogenesis aimed at retinal cell turnover throughout life., ((c) 2008 Wiley-Liss, Inc.)

Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.

Mammalian binocular vision relies on the divergence of retinal ganglion cell axons at the optic chiasm, with strictly controlled numbers projecting contralaterally and ipsilaterally. In mouse, contralateral projections arise from the entire retina, whereas ipsilateral projections arise from ventrotemporal retina. We investigate how development of these patterns of projection is regulated by the contralateral determinant Foxg1, a forkhead box transcription factor expressed in nasal retina and at the chiasm. In nasal retina, loss of Foxg1 causes increased numbers of ipsilateral projections and ectopic expression of the ipsilateral determinants Zic2, Ephb1 and Foxd1, indicating that nasal retina is competent to express an ipsilateral program that is normally suppressed by Foxg1. Using co-cultures that combine Foxg1-expressing with Foxg1-null retinal explants and chiasm cells, we provide functional evidence that Foxg1 promotes contralateral projections through actions in nasal retina, and that in chiasm cells, Foxg1 is required for the generation of a hitherto unrecognized activity supporting RGC axon growth.