Vessel Evaluation in Patients with Primary Open-Angle Glaucoma, Normal Tension Glaucoma and Healthy Controls.

PURPOSE: To compare changes in central retinal arterial equivalent (CRAE), central retinal vein equivalent (CRVE), arteriovenous ratio (AVR), tortuosity and fractal dimension in primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG) and in a control group (CG) on fundus photographs. Further, to provide further evidence of vascular change in glaucoma patients using a novel method of tortuosity. PATIENTS AND METHODS: The primary endpoint was the change in CRAE, CRVE, AVR, fractal dimension and tortuosity of the retinal vasculature from baseline, retrospectively analyzed from 2011 to 2017 at the University Eye Hospital Tuebingen. Fundus photos of POAG (N = 49), NTG (N = 38) and CG (N = 18) were computer evaluated and analyzed in the quantities mentioned above. RESULTS: CRAE in NTG and POAG and CRVE in NTG significantly decreased (P = 0.02, P = 0.01; P = 0.03) whereas CRVE in POAG increased insignificantly (P = 0.72). In NTG, AVR decreased significantly (P = 0.05), but to a lesser extent than in POAG (P < 0.001). In CG, CRAE decreased insignificantly (P = 0.10), CRVE decreased significantly (P = 0.03) and AVR increased insignificantly (P = 0.77). In POAG tortuosity calculated using standard methods as well as our novel method, increased significantly (P = 0.015-0.04), whereas it did not occur in NTG (P = 0.18-0.57) and CG (P = 0.11-0.21). Fractal dimensions in POAG decreased significantly (P = 0.001-0.002), whereas in NTG and CG changes were insignificant (P = 0.33-0.92). CONCLUSION: Based on a retrospective analysis of fundus photographs, specific retinal vasculature features of the retinal vasculature display significant alterations associated with NTG and POAG. The assessment of tortuosity using our novel method was consistent with previously established methods for analyzing tortuosity.

Electrical activation of degenerated photoreceptors in blind mouse retina elicited network-mediated responses in different types of ganglion cells.

Electrical (e-) stimulation is explored in schemes to rescue the vision of blind people, e.g. those affected by Retinitis Pigmentosa (RP). We e-activated subretinally the surviving degenerated photoreceptors (d-Phrs) of the rd1 mouse (RP model) and evoked visual responses in the blind retina. The e-stimulation was applied with a single platinum/iridium electrode. The d-Phrs (calcium-imaging) and ganglion cells (GC) activity (MEA-recording) were recorded in simultaneous multilayer recordings. The findings of this study confirm that the d-Phrs responded to e-stimulation and modulated the retinal network-activity. The application of blockers revealed that the synaptic interactions were dependent on voltage-gated calcium channels and mediated by the transmitters glutamate and GABA. Moreover, the gap junctions coupled networks promoted the lateral-spread of the e-evoked activity in the outer (~60 µm) and inner (~120 µm) retina. The activated GCs were identified as subtypes of the ON, OFF and ON-OFF classes. In conclusion, d-Phrs are the ideal interface partners for implants to elicit enhanced visual responses at higher temporal and spatial resolution. Furthermore, the retina's intact circuity at the onset of complete blindness makes it a tempting target when considering the implantation of implants into young patients to provide a seamless transition from blinding to chip-aided vision.

Feasibility study for a glutamate driven subretinal prosthesis: local subretinal application of glutamate on blind retina evoke network-mediated responses in different types of ganglion cells.

OBJECTIVE: A feasibility study for a transmitter based subretinal prosthesis, generating visual responses in blind mouse retina is presented. APPROACH: Degenerated rd1 mouse retina were stimulated in subretinal configuration by local glutamate (Glu) or NMDA application via micropipettes (~1.5 µm) and thereby the outer retinal activity was recorded by calcium-imaging or the ganglion cell (GC) activity was recorded by the multi-electrode array system. The network mediated activation of GC via bipolar cells was approved by the administration of Glu receptor blockers. MAIN RESULTS: Data of the degenerated and blind rd1 mouse retina reveals that the outer retina is Glu sensitive and that the subretinal Glu stimulation promotes network mediated GC responses. Analysis of the spatial activity-spread indicates that the Glu induced cell activation radius in the outer retina (~12.5 µm) and postsynaptically activated GC (~40 µm) is focal to the stimulation pipette tip. Moreover, the application of NMDA in subretinal space also evoked network mediated GC responses. The Glu-activated GC were identified as ON-OFF, OFF and two ON cells types. SIGNIFICANCE: This study evaluates the prerequisite for the function of a transmitter based implant, that after the loss of the photoreceptors, the remnant blind retinal network is Glu sensitive and functional, positively. The differential activation of ON (hyperpolarisation) and OFF (depolarisation) bipolar cells by transmitter Glu is a unique feature and of high interest for retinal implants. Therefore, the respective bipolar cell types could only be driven by glutamatergic stimulation accurately and not by electrical stimulation. The preserved functionality of the blind retina at the onset of complete blindness is motivating to continue research on a transmitter-based prosthesis. Since the artificial Glu stimulation mimics the natural retinal input, early implantation of a Glu-prosthesis might delay the devastating retinal remodelling positively, due to the neuronal-plasticity.

Rods progressively escape saturation to drive visual responses in daylight conditions.

Rod and cone photoreceptors support vision across large light intensity ranges. Rods, active under dim illumination, are thought to saturate at higher (photopic) irradiances. The extent of rod saturation is not well defined; some studies report rod activity well into the photopic range. Using electrophysiological recordings from retina and dorsal lateral geniculate nucleus of cone-deficient and visually intact mice, we describe stimulus and physiological factors that influence photopic rod-driven responses. We find that rod contrast sensitivity is initially strongly reduced at high irradiances, but progressively recovers to allow responses to moderate contrast stimuli. Surprisingly, rods recover faster at higher light levels. A model of rod phototransduction suggests that phototransduction gain adjustments and bleaching adaptation underlie rod recovery. Consistently, exogenous chromophore reduces rod responses at bright background. Thus, bleaching adaptation renders mouse rods responsive to modest contrast at any irradiance. Paradoxically, raising irradiance across the photopic range increases the robustness of rod responses.

Quality and learning curve of handheld versus stand-alone non-mydriatic cameras.

PURPOSE: Nowadays, complex digital imaging systems allow detailed retinal imaging without dilating patients' pupils. These so-called non-mydriatic cameras have advantages in common circumstances (eg, for screening or emergency purposes) but present limitations in terms of image quality and field of view. We compare the usefulness of two non-mydriatic camera systems (ie, a handheld versus a stand-alone device) for fundus imaging. The primary outcome was image quality. The secondary outcomes were learning effects and quality grade-influencing factors. METHODS: The imaging procedures followed standard protocol and were all performed by the same investigator. Camera 1 (DRS®) was a stand-alone system, while Camera 2 (Smartscope® PRO) was a mobile system. In order to evaluate possible learning effects, we selected an examiner with no prior training in the use of these systems. The images were graded separately by two experienced and "blinded" ophthalmologists following a defined protocol. RESULTS: In total, 211 people were enrolled. Quality grade comparisons showed significantly better grades for Camera 1. Both systems achieved better quality grades for macular images than for disc-centered images. No remarkable learning effects could be demonstrated. CONCLUSIONS: Both camera systems are useful for fundus imaging. The greater mobility of Camera 2 was associated with lower image quality. For screening scenarios or telemedicine, it must be determined whether image quality or mobility is more important.

Design, Implementation and Operation of a Reading Center Platform for Clinical Studies.

Clinical reading centers provide expertise for consistent, centralized analysis of medical data gathered in a distributed context. Accordingly, appropriate software solutions are required for the involved communication and data management processes. In this work, an analysis of general requirements and essential architectural and software design considerations for reading center information systems is provided. The identified patterns have been applied to the implementation of the reading center platform which is currently operated at the Center of Ophthalmology of the University Hospital of Tübingen.

Human Vision-Motivated Algorithm Allows Consistent Retinal Vessel Classification Based on Local Color Contrast for Advancing General Diagnostic Exams.

PURPOSE: Abnormalities of blood vessel anatomy, morphology, and ratio can serve as important diagnostic markers for retinal diseases such as AMD or diabetic retinopathy. Large cohort studies demand automated and quantitative image analysis of vascular abnormalities. Therefore, we developed an analytical software tool to enable automated standardized classification of blood vessels supporting clinical reading. METHODS: A dataset of 61 images was collected from a total of 33 women and 8 men with a median age of 38 years. The pupils were not dilated, and images were taken after dark adaption. In contrast to current methods in which classification is based on vessel profile intensity averages, and similar to human vision, local color contrast was chosen as a discriminator to allow artery vein discrimination and arterial-venous ratio (AVR) calculation without vessel tracking. RESULTS: With 83% ± 1 standard error of the mean for our dataset, we achieved best classification for weighted lightness information from a combination of the red, green, and blue channels. Tested on an independent dataset, our method reached 89% correct classification, which, when benchmarked against conventional ophthalmologic classification, shows significantly improved classification scores. CONCLUSIONS: Our study demonstrates that vessel classification based on local color contrast can cope with inter- or intraimage lightness variability and allows consistent AVR calculation. We offer an open-source implementation of this method upon request, which can be integrated into existing tool sets and applied to general diagnostic exams.

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2.

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.

Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.

Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.

Inferential testing for linkage with GENEHUNTER-MODSCORE: the impact of the pedigree structure on the null distribution of multipoint MOD scores.

The asymptotic distribution of [MOD] scores under the null hypothesis of no linkage is only known for affected sib pairs and other types of affected relative pairs. We have extended the GENEHUNTER-MODSCORE program to allow for simulations under the null hypothesis of no linkage to determine the empirical significance of MOD-score results in general situations. We performed simulations with families of different size (one million replicates of 500 families per simulation setting) to thoroughly investigate the impact of the pedigree size on the null distribution of multipoint MOD scores. It is shown that the distribution is dependent on the size and structure of the pedigrees under study. By performing simulations in the context of MOD-score analysis, our new tool efficiently explores the linkage data in a comprehensive way and also provides a valid method to inferentially test for linkage.

Linkage analysis using sex-specific recombination fractions with GENEHUNTER-MODSCORE.

MOTIVATION: Sex-specific marker maps have become increasingly available. We have implemented the usage of sex-specific recombination frequencies in the GENEHUNTER-MODSCORE program that performs multipoint linkage analysis. Furthermore, we have devised a consistent method to choose the combinations of male and female genetic positions at which linkage scores should be calculated. Marker coordinates can be read automatically from publicly available genetic maps. RESULTS: In a MOD-score analysis of the COGA dataset provided for Genetic Analysis Workshop 14, the highest linkage peak on chromosome 1 further increases when using sex-specific maps, while some smaller peaks are decreased. Simulations confirm that the MOD score can be biased when a sex-averaged instead of the correct sex-specific map is employed. This shows that an adequate modeling of the female:male ratio of genetic distances is important, especially for complex traits. AVAILABILITY: The new version of GENEHUNTER-MODSCORE can be downloaded from the following website: http://www.staff.uni-marburg.de/~strauchk/software.html

Linkage analysis of alcohol dependence using MOD scores.

Alcohol dependence is a typical example of a complex trait that is governed by several genes and for which the mode of inheritance is unknown. We analyzed the microsatellite markers and the Affymetrix single-nucleotide polymorphisms (SNPs) for a subset of the Collaborative Study on the Genetics of Alcoholism family sample, 93 pedigrees of Caucasian ancestry comprising 919 persons, 390 of whom are affected according to DSM III-R and Feighner criteria. In particular, we performed parametric single-marker linkage analysis using MLINK of the LINKAGE package (for the microsatellite data), as well as multipoint MOD-score analysis with GENEHUNTER-MODSCORE (for the microsatellite and SNP data). By use of two liability classes, different penetrances were assigned to males and females. In order to investigate parent-of-origin effects, we calculated MOD scores under trait models with and without imprinting. In addition, for the microsatellite data, the MOD-score analysis was performed with sex-averaged as well as sex-specific maps. The highest linkage peaks were obtained on chromosomes 1, 2, 7, 10, 12, 13, 15, and 21. There was evidence for paternal imprinting at the loci on chromosomes 2, 10, 12, 13, 15, and 21. A tendency to maternal imprinting was observed at two loci on chromosome 7. Our findings underscore the fact that an adequate modeling of the genotype-phenotype relation is crucial for the genetic mapping of a complex trait.

Efficient two-trait-locus linkage analysis through program optimization and parallelization: application to hypercholesterolemia.

We have optimized and parallelized the GENEHUNTER-TWOLOCUS program that allows to perform linkage analysis with two trait loci in the multimarker context. The optimization of the serial program, before parallelization, results in a speedup of a factor of more than 10. The parallelization affects the two-locus-score calculation, which is predominant in terms of computation time. We obtain perfect speedup, that is, the computation time decreases exactly by a factor of the number of processors. In addition, two-locus LOD and NPL scores are now calculated for varying genetic positions of both disease loci, not just one locus varied and the position of the other disease locus fixed, as before. This results in easily interpretable 3-D plots. We have reanalyzed a pedigree with hypercholesterolemia using our new version of GENEHUNTER-TWOLOCUS. Whereas originally, two individuals had to be discarded due to excessive computation-time demands, the entire 17-bit pedigree could now be analyzed as a whole. We obtain a two-trait-locus LOD score of 5.49 under a multiplicative model, compared to LOD scores of 3.08 and 2.87 under a heterogeneity and additive model, respectively. This further increases evidence for linkage to both 1p36.1-p35 and 13q22-q32 regions, and corroborates the hypothesis that the two genes act in a multiplicative way on LDL cholesterol level. Furthermore, we compare the computation times for two-trait-locus analysis needed by the programs GENEHUNTER-TWOLOCUS, TLINKAGE, and SUPERLINK. Altogether, our algorithmic improvements of GENEHUNTER-TWOLOCUS allow researchers to analyze complex diseases under realistic two-trait-locus models with pedigrees of reasonable size and using many markers.

Quantitative trait linkage analysis of longitudinal change in body weight.

One of the great strengths of the Framingham Heart Study data, provided for the Genetic Analysis Workshop 13, is the long-term survey of phenotypic data. We used this unique data to create new phenotypes representing the pattern of longitudinal change of the provided phenotypes, especially systolic blood pressure and body weight. We performed a linear regression of body weight and systolic blood pressure on age and took the slopes as new phenotypes for quantitative trait linkage analysis using the SOLAR package. There was no evidence for heritability of systolic blood pressure change. Heritability was estimated as 0.15 for adult life "body weight change", measured as the regression slope, and "body weight gain" (including only individuals with a positive regression slope), and as 0.22 for body weight "change up to 50" (regression slope of weight on age up to an age of 50). With multipoint analysis, two regions on the long arm of chromosome 8 showed the highest LOD scores of 1.6 at 152 cM for "body weight change" and of >1.9 around location 102 cM for "body weight gain" and "change up to 50". The latter two LOD scores almost reach the threshold for suggestive linkage. We conclude that the chromosome 8 region may harbor a gene acting on long-term body weight regulation, thereby contributing to the development of the metabolic syndrome.