Proteomics unravels the regulatory mechanisms in human tears following acute renouncement of contact lens use : a comparison between hard and soft lenses

Contact lenses (CLs) provide a superior alternative to spectacles. Although beneficial, the global burden of ocular dysfunctions attributed to regular use of CLs remains a topic of much challenge in ophthalmic research owing to debilitating clinical repercussions on the ocular surface, which are often manifested as breach in tear film integrity. This study elucidated the intricate tear proteome changes attributed to the use of different CLs (hard and soft) and unravelled, for the first time, the restorative mechanisms of several protein clusters following acute renouncement of CL use employing the label-free mass spectrometry-based quantitative proteomics approach. The expression patterns of certain proteins clusters were specific to the use of a particular lens type and a large majority of these actively regulates cell death and survival and, modulates cellular movement on the ocular surface. Noteworthy, CL use also evoked a significant upregulation of glycolytic enzymes associated with hypoxia and corresponding cognate metabolic pathways, particularly glucose metabolism and FXR/RXR pathways. Importantly, the assessment of CL renouncement unravelled the restorative properties of several clusters of proteins involved mainly in organismal injury and abnormalities and, cellular function and maintenance. These proteins play key roles in restoring tear homeostasis and wound-healing mechanisms post-CL use-elicited injury

Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future

Purpose Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. Design Meta-analysis of prevalence data. Participants A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. Methods AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). Main Outcome Measures Prevalence of early and late AMD, BCVA, and number of AMD cases. Results Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%–5.0%) in those aged 55–59 years to 17.6% (95%

First insight into the proteome landscape of the porcine short posterior ciliary arteries : key signalling pathways maintaining physiologic functions

Short posterior ciliary arteries (sPCA) provide the major blood supply to the optic nerve head. Emerging evidence has linked structural and functional anomalies of sPCA to the pathogenesis of several ocular disorders that cause varying degrees of visual loss, particularly anterior ischaemic optic neuropathy and glaucoma. Although the functional relevance of this vascular bed is well-recognized, the proteome of sPCA remains uncharacterized. Since the porcine ocular system closely resembles that of the human’s and is increasingly employed in translational ophthalmic research, this study characterized the proteome of porcine sPCA employing the mass spectrometry-based proteomics strategy. A total of 1742 proteins and 10527 peptides were identified in the porcine sPCA. The major biological processes involved in the maintenance of physiological functions of the sPCA included redox and metabolic processes, and cytoskeleton organization. These proteins were further clustered into diverse signalling pathways that regulate vasoactivity of sPCA, namely the tight junction, α- and β-adrenoceptor, 14-3-3, nitric oxide synthase and endothelin-1 -mediated signalling pathways. This study provides the first insight into the complex mechanisms dictating the vast protein repertoire in normal vascular physiology of the porcine sPCA. It is envisioned that our findings will serve as important benchmarks for future studies of sPCA

The gatekeepers in the mouse ophthalmic artery: endothelium-dependent mechanisms of cholinergic vasodilation

Cholinergic regulation of arterial luminal diameter involves intricate network of intercellular communication between the endothelial and smooth muscle cells that is highly dependent on the molecular mediators released by the endothelium. Albeit the well-recognized contribution of nitric oxide (NO) towards vasodilation, the identity of compensatory mechanisms that maintain vasomotor tone when NO synthesis is deranged remain largely unknown in the ophthalmic artery. This is the first study to identify the vasodilatory signalling mechanisms of the ophthalmic artery employing wild type mice. Acetylcholine (ACh)-induced vasodilation was only partially attenuated when NO synthesis was inhibited. Intriguingly, the combined blocking of cytochrome P450 oxygenase (CYP450) and lipoxygenase (LOX), as well as CYP450 and gap junctions, abolished vasodilation; demonstrating that the key compensatory mechanisms comprise arachidonic acid metabolites which, work in concert with gap junctions for downstream signal transmission. Furthermore, the voltage-gated potassium ion channel, Kv1.6, was functionally relevant in mediating vasodilation. Its localization was found exclusively in the smooth muscle. In conclusion, ACh-induced vasodilation of mouse ophthalmic artery is mediated in part by NO and predominantly via arachidonic acid metabolites, with active involvement of gap junctions. Particularly, the Kv1.6 channel represents an attractive therapeutic target in ophthalmopathologies when NO synthesis is compromised

gamma-Synuclein antibodies have neuroprotective potential on neuroretinal cells via proteins of the mitochondrial apoptosis pathway

The family of synuclein proteins (alpha, beta and gamma) are related to neurodegenerative disease e.g. Parkinson disease and Morbus Alzheimer. Additionally, a connection between gamma-synuclein and glaucoma, a neurodegenerative disease characterized by a progressive loss of retinal ganglion cells, which finally leads to blindness, exists. The reason for the development of glaucoma is still unknown. Recent studies evaluating the participation of immunological components, demonstrate complex changed antibody reactivities in glaucoma patients in comparison to healthy people, showing not only up-regulations (e.g. alpha-fodrin antibody) but also down-regulations (e.g. gamma-synuclein antibody) of antibodies in glaucoma patients. Up-regulated antibodies could be auto-aggressive, but the role of down-regulated antibodies is still unclear. Previous studies show a significant influence of the serum and the antibodies of glaucoma patients on protein expression profiles of neuroretinal cells. The aim of this study was to investigate the effect of gamma-synuclein antibody on the viability and reactive oxygen species levels of a neuroretinal cell line (RGC-5) as well as their interaction with cellular proteins. We found a protective effect of gamma-synuclein antibody resulting in an increased viability (up to 15%) and decreased reactive oxygen species levels (up to -12%) of glutamate and oxidative stressed RGC-5. These can be traced back to anti-apoptotic altered protein expressions in the mitochondrial apoptosis pathway indicated by mass spectrometry and validated by microarray analysis such as active caspase 3, bcl-2 associated-x-protein, S100A4, voltage-dependent anion channel, extracellular-signal-regulated-kinase (down-regulated) and baculoviral IAP repeat-containing protein 6, phosphorylated extracellular-signal-regulated-kinase (up-regulated). These changed protein expression are triggered by the gamma-synuclein antibody internalization of RGC-5 we could see in immunohistochemical stainings. These findings let us assume a novel physiological function of gamma-synuclein antibodies and give insights in the role of autoantibodies in glaucoma. We hypothesize that the down-regulation of autoantibodies found in glaucoma patients lead to a loss of protective autoimmunity

Effective melanin depigmentation of human and murine ocular tissues : an improved method for paraffin and frozen sections

PURPOSE: The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. METHODS: Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. RESULTS: Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65 degrees C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. CONCLUSIONS: Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues

Revision of encapsulated blebs after trabeculectomy : long-term comparison of standard bleb needling and modified needling procedure combined with transconjunctival scleral flap sutures

Purpose To compare two surgical approaches for treating encapsulated blebs after trabeculectomy with mitomycin C, in terms of the development of intraocular pressure and progression of glaucoma in a long-term follow up: 1. bleb needling alone vs. 2. a combined approach of needling with additional transconjunctival scleral flap sutures, to prevent early ocular hypotony. Methods Forty-six patients with failing blebs after trabeculectomy with mitomycin C were enrolled in this study. Patients received either needling revision alone (group 1; n = 23) or a combined needling with additional transconjuctival flap sutures, if intraoperatively the intraocular pressure was estimated to be low (group 2; n = 23). Intraocular pressure (IOP), visual acuity, visual fields, and optic nerve head configuration by means of Heidelberg Retina Tomograph (HRT®) were analysed over time. Results from both groups were compared using Mann-Whitney U-test for single timepoints. Results IOP did not differ significantly between the two groups during follow-up at three months (P = 0.13), six months (P = 0.12), one year (P = 0.92) and two years (P = 0.57) after surgery. Furthermore, there was no significant difference in the course of glaucoma concerning the optic nerve anatomy between the two groups (Rim Area Change in the Moorfields Regression Analysis of HRT®) till two years after surgery (P = 0.289). No functional impairment in visual acuity and visual fields was found in the groups of the study. Conclusions Single needling procedure is a standard successful method for restoring the function of encapsulated blebs. Postoperative hypotony represents a possible hazard, which can be minimized by additional transconjunctival flap sutures. Long-term results suggest that this modification is equally effective in lowering the IOP and preventing the progression of glaucoma as the standard needling procedure. To our knowledge this is the first study to investigate the long-term effect of tranconjunctival sutures for the prevention of hypotony

An in-depth view of the porcine trabecular meshwork proteome

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells