[Unmet research and developmental needs in ophthalmology : A consensus-based road map of the European Vision Institute for 2019-2025]. / Forschungs- und Entwicklungsbedarf in der Augenheilkunde ("Unmet needs") : Ein konsensbasierter Fahrplan des European Vision Institute (EVI) für die Jahre 2019 bis 2025.

PURPOSE: To define unmet needs in ophthalmology which can realistically be addressed in the next years (2019-2025) and to describe potential avenues for research to address these challenges. METHODS: Outcomes of a consensus process within the European Vision Institute (EVI, Brussels) are outlined. Disease areas which are discussed comprise glaucoma, retinal dystrophies, diabetic retinopathy, dry eye disease, corneal diseases, cataract and refractive surgery. RESULTS: Unmet needs in the mentioned disease areas are discussed and realistically achievable research projects outlined. CONCLUSION: Considerable progress can be made in the field of ophthalmology and patient-relevant outcomes in the near future.

[Integrated approach to the promotion of young academics in vision research at a European level]. / Integrativer Ansatz zur Nachwuchsförderung in der Sehforschung auf europäischer Ebene.

The European Commission predicts a dramatic dearth of researchers and doctors in the near future. At the same time, highly qualified and motivated human resources form the only guarantee for further development of scientific knowledge for research and clinical application. This situation calls for an integrated approach to the promotion of young academics in vision research at a European level. The Marie Curie Program of the European Union is an ideal tool, which can be used particularly efficiently in vision research to promote international networking and dedicated advancement of young academics. The exemplary chances and opportunities of this strategy can be demonstrated by six specific measures of the University Eye Hospital in Tübingen. In particular, strictly defined medical areas will need to fully exploit their innovation potential in the future in order to secure their position in the global research area or even to expand it. New organizational concepts and long-term career options as well as a clear commitment to cutting-edge performance are the prerequisites for effective promotion of young academics.

[The European Vision Institute. Opening up new frontiers?]. / Das "European Vision Institute". Aufbruch zu neuen Ufern?

The European Vision Institute EEIG (EVI) creates a new legal entity based on Community law to facilitate and encourage cross-border co-operation in vision research. Its major objectives are to conduct and support research, training, health information dissemination and other programmes with respect to blinding eye diseases, visual disorders, mechanisms of visual function, preservation of sight and the special health problems and requirements of the blind and visually disabled. EVI aims to foster centres in the EU in capacity building for innovative projects, to increase the flexibility, attractiveness and competitiveness of research careers, especially for young researchers. In addition, EVI will serve to co-ordinate activities with patient organisations and to build a pan-European platform for clinical trials.

[Establishing ophthalmology in the research framework programs of the European Union]. / Verankerung der Augenheilkunde in den Forschungsrahmenprogrammen der Europäischen Union.

The framework programmes (FP) of the European Commission have substantially contributed to the funding of research within the European countries. The contribution of the funding provided by the EU relative to the funding available on the national level has steadily increased. European ophthalmology and vision research has benefited from this support provided by the EU. This review introduces the European funding policies and the European Research Area (ERA) and provides a list of all projects in ophthalmology and vision research that have been funded within FP1 to FP6. As an example for new instruments within FP6, Integrated Projects, the EVI-GENORET project is introduced. Finally an outlook for FP7 is provided.

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus.

Infection with the neurotropic Borna disease virus (BDV) causes an immune-mediated neurological disease in a broad range of species. In addition to encephalitis, BDV-infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP-induced changes of intracellular Ca(2+)-concentration ([Ca(2+)](i)) in Müller cells from BDV-infected rat retinae. Freshly isolated cells were used for whole-cell patch-clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV-infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K(+) current densities. Müller cells from BDV-infected rats were characterized by expression of a prominent fast-inactivating A-type K(+) current which was rarely found in control cells. Moreover, the number of cells displaying Na(+) currents was slightly increased after BDV-infection. ATP evoked increases in [Ca(2+)](i) in Müller cells within retinal wholemounts of both control and BDV-infected animals. However, the number of ATP-responding isolated cells increased from 24% (age-matched controls) to 78% (cells from animals > or =18 weeks after infection). We conclude that in BDV-induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit.

Identification of purinergic receptors in retinal ganglion cells.

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC-RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X(3-5), P2X(7)). Immunohistochemistry with P2X(3,4) receptor subunit-specific antibodies showed a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

Expression of angiotensin-converting enzyme (ACE) in the developing chicken retina.

Angiotensin-converting enzyme (ACE) performs two contrasting enzymatic effects: as part of the renin-angiotensin system it converts angiotensin I into physiologically active angiotensin II, and it inactivates a number of peptides, e.g. substance P. These peptides are well known neurotransmitters in the retina and recently angiotensin II was described in retinal neurons. We therefore investigated a possible involvement of ACE in retinal metabolism by determining the mRNA and protein expression of ACE in the developing and mature chicken retina. ACE-mRNA expression was investigated by RT-PCR in the iris/ciliary body, the choroid, the optic nerve head, pecten, and the retina. Levels of ACE-mRNA were quantified by competitive PCR with heterologous competitor fragments in the retina at different developmental stages. To localize protein expression of ACE in the mature chicken retina an antibody directed against ACE was used. ACE-mRNA was present in all ocular tissues examined. Quantification of ACE-mRNA in avascular retinas of developing chickens revealed small amounts (0.13 attomol microl(-1)) at embryonic day 7 and values of about 0.6 attomol microl(-1)during embryonic days 7-17. ACE-mRNA expression transiently increased ten-fold (7.3 attomol microl(-1)) on postnatal day 1, decreased again to about 1.4 attomol microl(-1)on postnatal day 6, and remained constant thereafter. ACE-immunohistochemistry revealed labeling of photoreceptors, bipolar cells, amacrine cells, and cells in the ganglion cell layer as well as of Müller glia. Our data show that ACE-mRNA is an intrinsic component of the retina and that ACE itself has a widespread but distinct cellular distribution. The transient high expression of ACE-mRNA directly after hatching indicate, that ACE may be involved in fine tuning the neuropeptidergic equipment of the retinal network during the initial phase of visual experience.

Evidence for P2X(3), P2X(4), P2X(5) but not for P2X(7) containing purinergic receptors in Müller cells of the rat retina.

P2X receptors are ligand-gated ion channels activated by ATP. They are expressed in a broad variety of tissues. To date, eight P2X receptor subunits (P2X(1)-P2X(7), P2XM) have been cloned. In spite of the considerable evidence of signaling by extracellular nucleotides in other sensory systems, only few studies have been undertaken in the retina. In earlier studies, we have demonstrated that there is mRNA expression of the P2X(2-5) and P2X(7) subunits in the rat retina. In the present study, molecular biological methods were used to investigate expression of P2X receptor mRNA in freshly isolated Müller cells (MCs) of the adult rat retina (Brown Norway). A total of 36 MCs was analyzed, employing the single-cell RT-PCR. A positive amplification signal of 11/14 for P2X(3)-mRNA, 5/10 for P2X(4)-mRNA, 3/10 for P2X(5)-mRNA and 0/8 for P2X(7)-mRNA was revealed. Additionally, the astroglial identity of the cells under studied was confirmed in 10 cases by simultaneous amplification of RT-PCR products of glutamine synthetase (GS)- and P2X-mRNA. We conclude that MCs of rat retina express ionotropic P2 receptors, which, in addition to other functions, may play a key role within the recently described long range calcium signaling and the fast direct glia-neuron interactions in the rat retina.

Expression of purinergic receptors in bipolar cells of the rat retina.

P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina (Brown Norway, P25) using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X(3), P2X(4) and P2X(5) but not for P2X(7) mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina.

Coexpression patterns of mGLuR mRNAs in rat retinal ganglion cells: a single-cell RT-PCR study.

PURPOSE: Eight different subunits of metabotropic glutamate receptors (mGluRs) are known to date. mGluRs have been linked to an extensive list of neuromodulatory effects, depending on which intracellular or membrane-bound effector system is activated. Activation of mGluRs can influence neuronal activity and can result in changes of intracellular Ca2+ homeostasis-that is, changes in factors that are known to be crucial for cellular differentiation and cell death. Because mGluRs are known in modulating both intracellular and intercellular activities, this study was designed to determine which types of mGluRs are coexpressed in a neuron and whether distinct coexpression patterns can be found that reflect the different physiological requirements of a neuron at different stages of development and to learn whether neuronal injury results in adaptive changes of mGluR expression. METHODS: Juvenile and adult rat retinal ganglion cells (RGCs) and adult RGCs after axotomy were analyzed for their gene expression pattern of mGluRs by single-cell reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Adult RGCs predominantly expressed one or two different mGluR mRNAs, whereas juvenile RGCs coexpressed two and more. mGluR3, -5, and -7 mRNAs were found more frequently in juvenile than in adult RGCs. mGluR6 was detected in juvenile RGCs in low abundance but never in adult RGCs. However, mGluR6 was expressed in adult RGCs after axotomy. mGluR1 and -7 were also found more frequently in axotomized RGCs than in the adult control group. CONCLUSIONS: All types of mGluR mRNAs are expressed in RGCs. This is in contrast with previous in situ hybridization and immunohistochemical studies in which expression of mGluR3, -5, and -6 was not reported. The expression of some mGluR mRNAs seems to be developmental, although no distinct copatterns were found.

Angiotensin II receptor subtype gene expression and cellular localization in the retina and non-neuronal ocular tissues of the rat.

In addition to its function as a peripheral hormone, angiotensin II (AngII) has been shown to act as a neuromodulator in various brain regions. AngII effects are mediated by two major AngII receptor subtypes, AT1 and AT2, and different AT1 receptor isoforms AT1A and AT1B are described in rat brains. The purpose of the present study was to analyse the expression pattern of AT receptors in different parts of the rat eye with special emphasis on the retina. Specific primers were constructed and the gene expression of AngII receptor subtypes was investigated by means of reverse transcription-polymerase chain reaction (RT-PCR). An antibody was used for cellular localization of AT1 receptor in the retina. AT2 receptor mRNA was localized by in situ hybridization (ISH). We examined the retinas of different developmental stages as well as non-neuronal ocular tissues, e.g. choroid and anterior uveal tract of rats (Brown Norway and Wistar strain), for the gene expression of AT receptors. Our results show that AT1A and AT2 mRNAs are expressed in rat choroid, iris/ciliary body and retinas, whereas AT1B mRNA is not expressed in the retina but in all other ocular tissues under investigation. AT1 receptor immunohistochemistry of the retina showed strong labelling in the ganglion cell layer (GCL), and some cells in the inner nuclear layer (INL), suggesting putative ganglion cell but also amacrine cell labelling. In the retina, ISH for AT2 mRNA revealed labelling in the GCL and a faint labelling in the inner nuclear layer. No AT2 ISH-signal was found in the other ocular tissues. These data suggest that there is a specific distribution pattern of AT receptors in rat ocular tissues, especially in the retina. The expression of AT receptors on retinal ganglion cells confirms the AngII action on these cell types and supports the role of AngII as a retinal neurotransmitter or neuromodulator.

Expression of the P2X7-receptor subunit in neurons of the rat retina.

Despite the considerable evidence of signaling by extracellular nucleotides in other sensory systems, few studies have been undertaken in the eye. Molecular and immunohistochemical methods were used to demonstrate the expression and cellular localization of the P2X7 receptor subunit in the retina and choroid. RT-PCR was used for the detection of P2X7 subunit mRNA in the rat of different postnatal developmental stages (P23-P210) and revealed the presence of P2X7-mRNA in the retina, but not in the choroid. In the adult rat retina, immunolabelling for P2X7 was detected in a number of cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), suggesting different types of amacrine cells and ganglion cells. These results demonstrate for the first time the expression of the P2X7 receptor in the mammalian retina and furthermore in distinct neuronal cell populations. Our data suggest that extracellular ATP may provide both neuromodulatory and trophic influences on visual processing.

Gene expression of the P2X receptors in the rat retina.

Molecular-biological methods were used to demonstrate the expression of six P2X receptor subunits (P2X1-P2X6) in retina and choroid. Despite the considerable evidence for signalling by extracellular nucleotides in other sensory systems, few studies have been undertaken in the eye. RT-PCR for the detection of P2X subunit mRNA in the rat of different postnatal developmental stages (P23-P210) revealed the presence of P2X2 and P2X4 mRNA in the retina and choroid; P2X3, and P2X5 were detected only in the retina. There was no evidence for P2X1 and P2X6 mRNA in the ocular tissue under investigation. Our data suggest that extracellular ATP may have influences on visual processing.

Angiotensin II in the rabbit retina.

We investigated a putative local angiotensin II (AngII) system in the rabbit retina by examining AngII contents in the retina, vitreous humor, and choroid by radioimmunoassays and AngII synthesis in the retina and choroid by detection of angiotensin converting enzyme (ACE) mRNA. An antibody directed against AngII was used to localize possible cellular sources of AngII in the retina. To enhance immunoreactivity and to further examine AngII metabolism, tissues were preincubated in medium containing either protease inhibitors (PI), PI together with the AngII-precursor AngI, or PI and AngII. In some experiments the conversion of AngI to AngII was blocked by an ACE inhibitor. AngII concentration in the vitreous humor was only about 10% of the plasma concentration; in the retina and the choroid, however, AngII concentrations were 10 and 86 times higher, respectively, than in the plasma. ACE mRNA was present in both retina and choroid. Immunohistochemistry for AngII revealed faintly labeled amacrine cells at the inner border of the inner nuclear layer of the retina. Preincubation with PI resulted in an enhanced immunoreaction and in the labeling of fibers in the inner and outer plexiform layer; Müller cells and their processes as well as ganglion cells were now stained as well but the specificity of ganglion cell staining remains questionable. The immunoreaction was further enhanced when AngI or AngII was added to the incubation medium, whereas labeling totally disappeared when the conversion of AngI to AngII was blocked. No immunoreactive cells were detected in the choroid. In conclusion, the synthesizing enzyme for AngII is expressed in the retina and a specific AngII concentration is maintained there; AngII is localized in distinct cell types and can be metabolized within these cells. These data point to a local retinal AngII system that is protected and independent of blood-borne AngII.