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To further extent our understanding of aquatic vision, we introduce a complete optical model of a goldfish eye, which comprises all important optical parameters for the first time. Especially a spherical gradient index structure for the crystalline lens was included, thus allowing a detailed analysis of image quality, regarding spot size, and wavelength dependent aberration. The simulation results show, that our realistic eye model generates a sufficient image quality, with a spot radius of 4.9 mm which is below the inter cone distance of 5.5 mm. Furthermore, we optically simulate potential mechanical processes of accommodation and compare the results with contradictory findings of previous experimental studies. The quantitative simulation of the accommodation capacity shows that the depth of field is strongly dependent on the resting position and becomes significantly smaller when shorter resting positions are assumed. That means, to enable an extended depth perception with high acuity for the goldfish an adaptive, lens shifting mechanism would be required. In addition, our model allows a clear prediction of the expected axial lens-shift, which is necessary to ensure a sufficient resolution over a large object range.

It has been shown that mammalian retinal glial (Müller) cells act as living optical fibers that guide the light through the retinal tissue to the photoreceptor cells (Agte et al., 2011; Franze et al., 2007). However, for nonmammalian species it is unclear whether Müller cells also improve the transretinal light transmission. Furthermore, for nonmammalian species there is a lack of ultrastructural data of the retinal cells, which, in general, delivers fundamental information of the retinal function, i.e. the vision of the species. A detailed study of the cellular ultrastructure provides a basic approach of the research. Thus, the aim of the present study was to investigate the retina of the spectacled caimans at electron and light microscopical levels to describe the structural features. For electron microscopy, we used a superfast microwave fixation procedure in order to achieve more precise ultrastructural information than common fixation techniques. As result, our detailed ultrastructural study of all retinal parts shows structural features which strongly indicate that the caiman retina is adapted to dim light and night vision. Various structural characteristics of Müller cells suppose that the Müller cell may increase the light intensity along the path of light through the neuroretina and, thus, increase the sensitivity of the scotopic vision of spectacled caimans. Müller cells traverse the whole thickness of the neuroretina and thus may guide the light from the inner retinal surface to the photoreceptor cell perikarya and the Müller cell microvilli between the photoreceptor segments. Thick Müller cell trunks/processes traverse the layers which contain light-scattering structures, i.e., nerve fibers and synapses. Large Müller cell somata run through the inner nuclear layer and contain flattened, elongated Müller cell nuclei which are arranged along the light path and, thus, may reduce the loss of the light intensity along the retinal light path. The oblique arrangement of many Müller cell trunks/processes in the inner plexiform layer and the large Müller cell somata in the inner nuclear layer may suggest that light guidance through Müller cells increases the visual sensitivity. Furthermore, an adaptation of the caiman retina to low light levels is strongly supported by detailed ultrastructural data of other retinal parts, e.g. by (i) the presence of a guanine-based retinal tapetum, (ii) the rod dominance of the retina, (iii) the presence of photoreceptor cell nuclei, which penetrate the outer limiting membrane, (iv) the relatively low densities of photoreceptor and neuronal cells which is compensated by (v) the presence of rods with long and thick outer segments, that may increase the probability of photon absorption. According to a cell number analysis, the central and temporal areas of the dorsal tapetal retina, which supports downward prey detection in darker water, are the sites of the highest diurnal contrast/color vision, i.e. cone vision and of the highest retinal light sensitivity, i.e. rod vision.

Background/ Aims: This study was performed to reveal signaling pathways exploited by pigment epithelium-derived factor (PEDF) derived from retinal (glial) Müller cells to protect retinal ganglion cells (RGCs) from cell death. Methods: The survival of RGCs was determined in the presence of conditioned culture media (MCM) from or in co-cultures with Müller cells. The significance of PEDF-induced STAT3 activation was evaluated in viability assays and using Western blotting analyses and siRNA-transfected cells. Results: Secreted mediators of Müller cells increased survival of RGCs under normoxia or hypoxia to a similar degree as of PEDF- or IL-6-exposed cells. PEDF and MCM induced an increased STAT3 activation in RGCs and R28 cells, and neutralization of PEDF in MCM attenuated STAT3 activation. Inhibition of STAT3 reduced PEDF-promoted survival of RGCs. Similar to IL-6, PEDF induced STAT3 activation, acting in a dose-dependent manner via the PEDF receptor (PEDF-R) encoded by the PNPLA2 gene. Ablation of PEDF-R attenuated MCM-induced STAT3 activation and compromised the viability of PEDF-exposed R28 cells. Conclusions: Müller cells are an important source of PEDF, which promotes RGC survival through STAT3 activation and, at least in part, via PEDF-R. Enhancing the secretory function of Müller cells may be useful to promote RGC survival in retinal neurodegenerative diseases.

A comprehensive schematic eye model of the hooded merganser is introduced for the first time to advance the understanding of amphibious vision. It is comprised of two different configurations, the first one modeling its visual system in air (unaccommodated state) and the second one representing the case where the eye is immersed in water (accommodated state). The model was designed using available data of former studies, image analysis and the implementation of feasible assumptions that serve as starting values. An optimization process incorporating an optical design program is used to vary the starting values with the aim of finding the setup offering the best acuity. The image quality was measured using the root-mean-square radius of the focal spot formed on the retina. The resulting schematic eye model comprises all relevant optical specifications, including aspherical geometrical parameters for cornea and lens, distances between the surfaces, the gradient index distribution of the lens, the retinal specifications and the object distance in both media. It achieves a spot radius of 4.20 μ m for the unaccommodated state, which meets the expectations derived by the mean ganglion cell density and comparison with other animals. In contrast, under water the determined spot radius of 11.48 µ m indicates an acuity loss. As well as enhancing our understanding of the vision of the hooded merganser, the schematic eye model may also serve as a simulation basis for examing similar animal eyes, such as the cormorant or other fish hunting birds.

Stroke-induced blood–brain barrier breakdown promotes complications like cerebral edema and hemorrhagic transformation, especially in association with therapeutical recanalization of occluded vessels. As arteries, capillaries and veins display distinct functional and morphological characteristics, we here investigated patterns of blood–brain barrier breakdown for each segment of the vascular tree in rodent models of embolic, permanent, and transient middle cerebral artery occlusion, added by analyses of human stroke tissue. Twenty-four hours after ischemia induction, loss of blood–brain barrier function towards FITC-albumin was equally observed for arteries, capillaries, and veins in rodent brains. Noteworthy, veins showed highest ratios of leaky vessels, whereas capillaries exhibited the most and arteries the least widespread perivascular tracer extravasation. In contrast, human autoptic stroke tissue exhibited pronounced extravasations of albumin around arteries and veins, while the pericapillary immunoreactivity appeared only faint. Although electron microscopy revealed comparable alterations of the arterial and capillary endothelium throughout the applied animal models, structural loss of arterial smooth muscle cells was only observed in the translationally relevant model of embolic middle cerebral artery occlusion. In light of the so far available concepts of stroke treatment, the consideration of a differential vascular pathophysiology along the cerebral vasculature is likely to allow development of novel effective treatment strategies.

Retinal Müller glial cells have been shown to undergo reactive gliosis in a variety of retinal diseases. Upregulation of glial fibrillary acidic protein (GFAP) is a hallmark of Müller cell activation. Reactive gliosis after retinal detachment or ischemia/reperfusion is characterized by hypertrophy and downregulation of inwardly rectifying K+ (Kir) currents. However, this kind of physiological alteration could not be detected in slowly progressing retinal degenerations. The photoreceptor toxin N-methyl-N-nitrosourea (MNU) leads to the rapid loss of cells in the outer nuclear layer and subsequent Müller cell activation. Here, we investigated whether Müller cells from MNU-treated mice exhibit reactive gliosis. We found that Müller cells showed increased GFAP expression and increased membrane capacitance, indicating hypertrophy. Membrane potential and Kir channel-mediated K+ currents were not significantly altered whereas Kir4.1 mRNA expression and Kir-mediated inward current densities were markedly decreased. This suggests that MNU-induced Müller cell gliosis is characterized by plasma membrane increase without alteration in the membrane content of Kir channels. Taken together, our findings show that Müller cells of MNU-treated mice are reactive and respond with a form of gliosis which is characterized by cellular hypertrophy but no changes in Kir current amplitudes.

A fovea, the retinal pitted invagination in the area centralis, is found in many vertebrates such as fish, reptiles, birds and in higher primates including humans. Because of the refractive index step between the retinal tissue and vitreous, the fovea defines an optically effective interface with aspherical characteristics, which influences the ray propagation to the receptor layer buried about 250 μm within the retinal layers [1]. In principle, two basic foveal shapes can be distinguished [2]. On the one hand, the convexiclivate type of fovea is a deep, funnel-shaped depression with a convex slope of the side walls and is observed in birds, some lizards, and certain deep-sea teleosts. On the other hand, the concaviclivate type of fovea is shallow and gradually shaped; the human fovea is of this type. Previous examinations on the influence of the fovea on the image-forming process have mainly been focused on the steep foveae types in which increasing magnification and directional-dependent distortion effects were predicted [3], [4], [5], [6], [7]. These investigations have been restricted to simple geometrical descriptions of the foveal shape, such as spheres and paraboloids. Real geometrical foveal structures from living creatures have not yet been examined. Moreover, the ray-trace considerations have been partly reduced to a few selected rays approaching the vitreo-retinal interface, and very limited numbers of incidence directions and wavelength-dependent effects have been investigated. Studies on the refracting characteristics of human foveae types are even more rare [2], [8]. C. Ross analyzed the optical functionality of the foveae of tarsiers, neglecting the influence of the anterior part of the eye and regarding only position shifts of three single rays. This contribution aims to answer the question on whether a comprehensive human eye model, which takes all optical components and their aberrations into account, leads to more detailed findings. In particular, field- and wavelength-dependent influences on spot sizes and positions, based on actual ray-bundle-cones are considered, which allow the estimation of aberration effects. With the current investigation, we address this issue by combining a realistic anatomical model of the anterior parts of the eye with real human fovea geometries. For the anterior eye, the human eye model of Liou and Brennan [9], with a gradient index profile of the crystalline lens, was used. To investigate the influence of actual fovea geometries on image formation, two selected and different foveal shapes (average and extraordinary), based on in vivo optical coherence tomography (OCT) measurements of a human eye were used. We analyzed the ray-optical behavior and implemented additional considerations that involve the variation of the observed field position, pupil diameter, chromatic effects, and especially the imaging behavior of peripheral colors in the receptor plane when the primary color is focused on this theoretical surface. These simulations validate and expand the knowledge about the correlation between aperture size and real and theoretical spot size, the effect of the foveal shape on either spot size and axial spot position, and, finally, the lateral spot position and color dependency of the incident angle.

Regulation of cellular volume is of great importance to avoid changes in neuronal excitability resulting from a decrease in the extracellular space volume. We compared the volume regulation of retinal glial (Müller) and neuronal (bipolar) cells under hypoosmotic and glutamate-stimulated conditions. Freshly isolated slices of the rat retina were superfused with a hypoosmotic solution (60% osmolarity; 4 min) or with a glutamate (1 mM)-containing isoosmotic solution (15 min), and the size changes of Müller and bipolar cell somata were recorded. Bipolar cell somata, but not Müller cell somata, swelled under hypoosmotic conditions and in the presence of glutamate. The hypoosmotic swelling of bipolar cell somata might be mediated by sodium flux into the cells, because it was not observed under extracellular sodium-free conditions, and was induced by activation of metabotropic glutamate receptors and sodium-dependent glutamate transporters. The glutamate-induced swelling of bipolar cell somata was mediated by sodium chloride flux into the cells induced by activation of NMDA- and non-NMDA glutamate receptors, glutamate transporters, and voltage-gated sodium channels. The glutamate-induced swelling of bipolar cell somata was abrogated by adenosine and γ-aminobutyric acid, but not by vascular endothelial growth factor and ATP. The data may suggest that Müller cells, in contrast to bipolar cells, possess endogenous mechanisms which tightly regulate the cellular volume in response to hypoosmolarity and prolonged glutamate exposure. Inhibitory retinal transmission may regulate the volume of bipolar cells, likely by inhibition of the excitatory action of glutamate.

Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Muller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Muller cell-RGC-co-cultures we observed that activity of Muller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Muller cells towards RGC.

Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca(2+) signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca(2+) -dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer's disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns.

Brain edema in acute hepatic encephalopathy (HE) is due mainly to swelling of astrocytes. Efflux of potassium is implicated in the prevention of glial swelling under hypoosmotic conditions. We investigated whether pathogenic factors of HE, glutamine (Gln) and/or ammonia, induce alterations in the expression of glial potassium channels (Kir4.1, Kir2.1) and Na(+) -K(+) -2Cl(-) cotransporter-1 (NKCC1) in rat cerebral cortex and cultured rat cortical astrocytes and whether these alterations have consequences for potassium efflux and astrocytic swelling. Thioacetamide-induced acute liver failure in rats resulted in significant decreases in the Kir4.1 mRNA and protein contents of cerebral cortex, whereas expression of Kir2.1 and NKCC1 remained unaltered. Incubation of primary cortical astrocytes for 72 hr in the presence of Gln (5 mM), but not of ammonia (5 mM or 10 mM), induced a decrease in the levels of Kir4.1 mRNA and protein. Similarly to incubation with Gln, reduction of Kir4.1 mRNA expression by RNA interference caused swelling of astrocytes as shown by confocal imaging followed by 3D computational analysis. Gln reduced the astrocytic uptake of D-[(3) H]aspartate, but, in contrast to the earlier reported effect of ammonia, this reduction was not accompanied by decreased expression of the astrocytic glutamate transporter GLT-1 mRNA. Both Gln and ammonia decreased hypoosmolarity-induced (86) Rb efflux from the cells, but the effect was more pronounced with Gln. The results indicate that down-regulation of Kir4.1 may mediate distinct aspects of Gln-induced astrocytic dysfunction in HE.

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

Diabetic retinopathy is characterized by pericyte loss and vasoregression both in the clinic and in animal models. A mild neurodegeneration with loss of ganglion cells is also described in the diabetic retina. Like VEGF-A, Epo is angioprotective and, in high doses, neuroprotective, however, without affecting vessel permeability. This study was to investigate the effect of a long-term suberythropoietic dose of Epo on vascular damage and neurodegeneration in a rat model of diabetic retinopathy.We administered Epo 3x256 IU/kg body weight/week to streptozotocin-diabetic Wistar rats for up to 6 months. Leukostasis was analyzed by quantitation of CD45 positive cells adherent to the retinal microvasculature. VEGF-A levels were assessed by Elisa at 3 months of treatment. Vasoregression was quantified in retinal digest preparations after 6 months of Epo treatment. Neurodegeneration was analyzed from PAS stained retinal paraffin preparations.Leukostasis was unaffected by treatment with Epo which significantly inhibited the loss of pericyte and the formation of acellular capillaries. Neurodegeneration in the diabetic retina was significantly reduced by Epo treatment. Increased VEGF-A levels in the diabetic retina were normalized by Epo treatment.Suberythropoietic Epo is effective to protect microvascular and neuronal damage in the experimental diabetic retina.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X2 and P2X5 receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3–1000 µm; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS; 30 µm) counteracted the ATP response. In contrast to ATP, α,β-meATP (30 µm; for 1 s) was virtually ineffective. Prolonged application of ATP (100 µm; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X2−2 splice variant-mediated responses. Using single-cell RT-PCR, P2X2, P2X2−2 and P2X5 mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X2 isoforms was not observed. Laser scanning microscopy revealed both P2X2 and P2X5 immunoreactivity in virtually every TH-positive neuron. P2X2 immunoreactivity was largely distributed over the cell body, whereas P2X5 immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X2, P2X2−2 and P2X5 receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X2/P2X2−2 compared with P2X5 suggest that the excitatory P2X responses are mediated by P2X2 and P2X2−2 receptors. Apparently there exist two types of P2X2 receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X2−2 splice variant.

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Muller glial cells normally mediate the osmohomeostasis in the inner retina (mainly via channel-mediated transport of potassium and water), we determined whether retinal light injury causes an alteration in the retinal localization of glial water (aquaporin-4) and potassium (Kir4.1) channels, and in the potassium conductance of Muller cells. Mice were treated with bright white light (intensity, 15,000 lx) for 2 h. Light treatment results in Muller cell gliosis as indicated by the enhanced staining of the glial fibrillary acidic protein and an increase in the cell membrane area reflecting cellular hypertrophy. In light-injured retinas, the immunostaining of the photoreceptor water channel aquaporin-1 disappeared along with the degeneration of the outer retina, and the outer nuclear layer contained large spherical bodies representing photoreceptor nuclei which were fused together. The immunostainings of the aquaporin-4 and Kir4.1 proteins were increased in the outer retina after light treatment. Since the amplitude of the potassium currents of Muller cells remained largely unaltered, the increase in the Kir4.1 immunostaining is supposed to be caused by a redistribution of the channel protein. The data indicate that Muller glial cells respond to excessive light with an alteration in the localization of Kir4.1 and aquaporin-4 proteins; this alteration is thought to be a response to the edema in the outer retina and may support the resolution of edema.

Muller cells, the principal glial cells of the retina of all vertebrates, were discovered already in 1851 but it was only in the past decades that it became evident that these cells are essential players in vision. Muller cells constitute the ‘core’ of columnar units of clonally and functionally related groups of neurons. Their primary function is to support neuronal functioning (by increasing the signal-to-noise ratio of information processing) and survival (by maintaining a metabolic ‘symbiosis’ with the neurons). It has been shown that Muller cells increase the signal-to-noise ratio of retinal information processing by, for instance, (i) guiding the light towards the photoreceptor cells, (ii) removing excess neurotransmitter molecules from extracellular space, and (iii) performing an efficient clearance of excess extracellular potassium ions after neuronal excitation. The latter two functions are also crucial for neuronal survival – to prevent excitotoxic effects of glutamate – and are coupled to water clearance which is equally important for neuronal survival. As another case of glial homeostasis, the maintenance of appropriate microenvironmental biomechanics has recently been described. Normal glial cells are softer than the neurons, and thus provide a suitable substrate for neurite growth during development, and for functional plasticity. Finally it should be pointed out that Muller cells are capable of ‘sensing’ neuronal activity. They respond to physiological light stimulation of their adjacent photoreceptors with two distinct types of intracellular calcium rises. These calcium rises then may trigger the release of signal substances, incl. so-called ‘gliotransmitters’, from Muller cells which thus even may modify neuronal signal processing in the retina (e.g., in cases of adaptation to bright light, as another mechanism to increase the signal-to-noise ratio of information processing in retina). In cases of reactive Muller cell gliosis, the dominant potassium conductance of the membrane is down-regulated and an increased expression of intermediate filaments is associated with increasing stiffness of the glial cell processes, such that all above-mentioned glial functions are impaired.

Objective: The current study introduced OCT as a novel tool in clinical veterinary ophthalmology in a much wider variety of free-living avian species than hitherto studied. Methods: OCT was tested and performed in 39 free-living birds (21 species of 12 families) and compared to direct ophthalmoscopy. Birds were examined with combinations of different restraints (manual restraint or fixation on a holding device) and different anaesthesia regimes (none, sedation, general anaesthesia). Inter- and intra-species specific variations of the general procedure, the restraint methods and the clinical findings were evaluated. Results: OCT was possible in all examined avian species (from 40 g up to 7720 g) and superseded direct ophthalmoscopy in quality and quantity of ophthalmological findings. All restraint methods enabled OCT examination, however combination of general anaesthesia and a holding device provided the most rapid and subjectively the least stressful examination technique. Stability, stress reduction, head angle and distance from the OCT device were important factors influencing volume scanning quality. Sixteen, out of 39 birds, presented ocular abnormalities detected by OCT (compared to only five birds when using direct ophthalmoscopy). OCT with included fundus images offered an objective assessment of retinal changes. Retinal abnormalities included changes of fundus pigmentation, drusenoid changes and severe retinal and choroidal degenerations. Species-specific variations of retinal layer dimensions and of foveal structures were evident. Conclusion: OCT is a promising, non-invasive method, which significantly compliments standard techniques. OCT is applicable to a wide variety of avian species; it provides high quality cross-sectional images of the retina, enabling accurate and improved diagnosis and prognosis of therapies. An assessment of the visual capabilities of traumatised birds is a major factor for their rehabilitation and survival in the wild. Finally, this method is an excellent tool in interdisciplinary retinal research, providing novel insights into the diversity of very specialized structural adaptations of avian retina.

Exposure of cultured cortical neurons to elevated extracellular K+ concentrations (25 mM) induces membrane depolarization and an increase in action-potential firing. Long-term high K+ treatment was associated with an increased neuronal cell death. In surviving neurons, multiple changes occurred in the proportion of individual NMDA receptor subunit 1 (NR1) splice variant mRNA expression, whereas the overall expression of NR1, NR2A and NR2B transcripts remained unaffected. The high K+-induced changes in NR1 splice variant expression were virtually abolished upon a concurrent administration of tetrodotoxin (TTX; 3 μM). In voltage-clamp recordings performed on neurons resistant to high K+ treatment, inward currents induced by NMDA (1–1000 μM) were reduced. In K+-resistant cells, the activity of calpain but not of caspase-3 was diminished compared with controls kept in regular medium. NR function as well as calpain activity was not affected in cultures concomitantly treated with high K+ and either TTX or a NR antagonist (CGS19755 (selfotel) or memantine). In conclusion, the present data indicate adaptive changes in NR1 splice variant expression and a decrease in NR function upon a sustained increase in neurotransmission. Accordingly, alternative splicing could be an endogenous mechanism to counteract cellular damage due to overactivation of excitatory NRs and may be associated with an impairment of necrotic mechanisms. British Journal of Pharmacology (2006) 147, 622–633. doi:10.1038/sj.bjp.0706471

Under normal conditions, the pigment epithelium dehydrates the outer retina while Muller glial cells mediate the rapid water transport within the inner retina. Gliotic alterations of Muller cells may be implicated in the development of oedema in the post-ischaemic retina. Here, we suggest a mechanism of Muller cell-supported neuronal cell swelling and apoptosis in the ischaemic retina. During ischaemia, over-excitation of ionotropic glutamate receptors leads to neuronal cell depolarization that causes excess Ca(2+) influx into the cells, and to activation of the apoptosis machinery. The ion fluxes into the retinal neurons are associated with water movements that are mediated by aquaporin-4 water channels expressed by Muller cells and result in neuronal cell swelling. After reperfusion, the glial cells may swell due to the down-regulation of their K(+) conductance, which results in intracellular K(+) overload and water movements from the blood and vitreous into the cells. An inhibition of the glial cell-mediated water movements during ischaemic episodes should reduce the ion shifts at the neuronal synapses, resulting in decreased neuronal cell swelling and apoptosis. An inhibition of the water movements in the post-ischaemic phase may prevent cytotoxic Muller cell swelling but may impair the fluid clearance from retinal tissue in the presence of vasogenic oedema. Thus, pharmacological modification of the ion and fluid clearance functions of Muller cells may become a novel way to resolve both cytotoxic and vasogenic oedema in the retina.

Studies in rodents suggest the presence of a hepatopancreatic stem cell in adult pancreas that may give rise to liver cells in vivo. The aim of the present study was to determine the ability of human islet-derived cells to adopt a hepatic phenotype in vivo. Cultured human islet-derived progenitor cells that did not express albumin in vitro were stained with the red fluorescent dye PKH26 and injected into the liver of severe combined immunodeficiency mice. After 3 or 12 weeks, red fluorescent cells were detected in 11 of 15 livers and were mostly single cells that were well integrated into the liver tissue. Human albumin was found in 8 of 11 animals by immunohistochemistry, and human albumin mRNA was detected in 4 of 10 host livers. The mechanism underlying this phenomenon seems to be transdifferentiation, because human and mouse albumin were found to be expressed in distinct cells in the host liver.

MPP4 and MPP5 are closely related members of the p55-subfamily of membrane-associated guanylate kinases (MAGUKs) known to mediate the assembly of protein complexes at the plasma membrane of cell-cell junctions. Both MPP4 and MPP5 have been implicated in retinal function; however, their specific roles in the cellular mechanisms underlying vision are largely unknown. Here, we generated specific poly- and monoclonal antibodies against the two proteins and show that MPP4 and MPP5 are localized at distinct sites of cell-cell contact in the mouse retina. While MPP4 is a component of the synaptic terminals of photoreceptors, MPP5 exclusively localizes to apical membrane domains of the outer limiting membrane (OLM) junctions. The vertebrate homologs of Caenorhabditis elegans lin-7, Veli1, -2, and -3, have previously been identified as putative binding partners of MPP5. In this study, we show that MPP4 directly interacts with the Veli proteins via L27 heterodimerization in vitro. In addition, two of the three Veli isoforms, Veli1 and -3, are demonstrated to be expressed in the mouse retina. Immunofluorescence microscopy reveals extensive colocalization of Veli3 with both MPP4 and MPP5. This association of Veli3 with either MPP4 or MPP5 suggests that the MAGUKs recruit Veli3 and its binding partners to different cellular regions of the retina where they may participate in the organization of specialized intercellular junctions.

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.

In epithelial and muscle cells, intermediate filaments (IFs) are important for resistance to mechanical stress. The aim of this study was to elucidate whether IFs are also important for providing resistance to mechanical stress in the Muller cells of the retina and whether this has any pathophysiological consequences. We used mice deficient in IF proteins glial fibrillary acidic protein and/or vimentin ( GFAP –/– , Vim –/– and GFAP –/– Vim –/– ), and stress on the retina was applied by excision of the eyes immediately post mortem (compared with in situ fixation) or by inducing a neovascular response to oxygen-induced retinopathy (OIR). The structure of unchallenged retinas was normal, but mechanical stress caused local separation of the inner limiting membrane (ILM) and adjacent tissue from the rest of the retina in GFAP –/– Vim –/– mice and, to a lesser extent, in Vim –/– mice. This detachment occurred within the endfeet of Muller cells, structures normally rich in IFs but IF-free in GFAP –/– Vim –/– mice. Hypoxia-induced neovascularization was comparable in all groups of mice with respect to the retinal surface area occupied by new vessels. However, the vessels traversed the ILM and penetrated the vitreous body less frequently than in wild-type retinas (31-55% in Vim –/– , 66-79% in GFAP –/– Vim –/– ). We conclude that IFs are important for maintaining the mechanical integrity of Muller-cell endfeet and the inner retinal layers under a mechanical challenge. Furthermore, the absence of IFs in Muller cells leads to an abnormal response of the vascular system to ischemia, specifically decreased ability of newly formed blood vessels to traverse the ILM.

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system—composed of sensory cells, neurons and radial glial cells—was probably the plesiomorphic condition in the bilaterian ancestor., C.H. was financed by a personal research fellowship from the DFG (HE 7224/1-1). A.K. was financially supported by the ‘Bundesministerium für Bildung und Forschung’ (BMBF 1315883), the DFG (SPP 175/) and the University of Leipzig Medical Faculty. S.K.-S. was funded by a Lise-Meitner grant from the Austrian Science Fond (FWF) (M 1485-B19) and C.B. is supported by a ‘Ramón y Cajal’ fellowship by the MINECO, project no. RYC-2014-15615.

Purpose High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA. Results Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. Conclusions Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

Purpose High myopia is characterised by excessive eye growth and mechanically weakened, thinned and stretched scleral tissue. Enhanced chemical cross linking of collagen fibrils in the sclera (SXL) is a promising therapeutic approach to stiffen the sclera and to prevent eye globe elongation. Therefore, it is important to understand the role of collagen fibril producing fibroblasts and the extracellular matrix remodelling enzymes in normal and myopic eyes and after SXL treatment. Methods We examined scleral tissue of normal rabbit eyes and after SXL treatment by means of histology and electron microscopy. We characterised expression profiles of cellular markers (e.g. Vimentin) and of relevant enzymes (e.g. MMPs) by means of RT-PCR analysis and immunohistochemistry in cell cultures from human and rabbit scleral tissue. Results We observed altered scleral collagen fibril profiles and activated scleral fibroblasts - signs of remodelling processes after SXL treatment. Fibroblasts are capable to produce and secrete collagen and different matrix-degrading and regulatory enzymes (e.g. MMP1-3 and 9; TIMP1-4; LOX). Conclusion Significant remodelling processes were induced after SXL treatment and the role of each specific contributor/enzyme has to be determined for an efficient SXL treatment. increases scleral stiffness. We want to investigate the influence of relevant enzymes on remodelling processes after SXL to optimize the new and promising therapeutic approach.

Purpose The application of riboflavin and blue light (for Collagen Crosslinking of scleral tissue; SXL) was proved as a possible therapeutic method to increase scleral stiffness and therefore, to inhibit eye growth in young rabbits. Methods For eye growth examination the sclera of young rabbit eyes were treated with riboflavin and 10 mW/cm² blue light. MR-Imaging and ultrasonic A scans were used to measure effects of SXL treatment on eye growth. Safety and efficacy parameters were determined using immunohistochemical and histological methods. Results Immunohistochemical and histological examinations showed no neurodegenerative side effects or signs of glial reactivity in retinal tissue beyond a light intensity of 400 mW/cm². Young animals treated with a minimal effective light energy dose of 10 mW/cm² displayed a significant reduction of their eye growth. The eye growth inhibition after SXL treatment maintained for at least 24 weeks. Conclusion The results confirm the idea that scleral collagen crosslinking by riboflavin/blue light is an applicable method to inhibit eye growth and therefore, it might be a treatment for pathologic myopia.

Purpose In our research project “collagen crosslinking of scleral tissue (SXL)”, the application of riboflavin and blue light has been proved as a possible therapeutic method to increase scleral stiffness. Nevertheless, it is necessary to develop a new application system applicable in human. Methods Rheology was used to investigate changes of sclera stiffness after SXL treatment. An adapted and modified blue light irradiations system has been developed to apply various blue light intensities. Results As a proof of principle, we demonstrate a dose dependent increase of scleral stiffness after application of various blue light energy dose rates. Increased biomechanical stiffness could be induced with light dose rates up to 200 mW/cm². However, the light application system used for animal surgery has serious limitations. The animal experiments revealed relevant technical parameters and helps to design a suitable substance and application system for usage in human. Conclusion The results confirm the idea that SXL by riboflavin and blue light irradiation is an applicable method to increase biomechanical stiffness of scleral tissue and therefore, it might be a treatment for pathologic myopia. For the therapeutic application in patients a special substance application and irradiation system has to be refined and developed further.

Ischemic stroke not only impairs neuronal function but also affects the cerebral vasculature as indicated by loss of blood—brain barrier (BBB) integrity. Therefore, therapeutical recanalization includes an enhanced risk for hemorrhagic transformation and bleeding, traditionally attributed to a ‘reperfusion injury’. To investigate the mechanisms underlying ischemia-/reperfusion-related BBB opening, we applied multiple immunofluorescence labeling and electron microscopy in a rat model of thromboembolic stroke as well as mouse models of permanent and transient focal cerebral ischemia. In these models, areas exhibiting BBB breakdown were identified by extravasation of intravenously administered fluorescein isothiocyanate (FITC)-albumin. After 24 hours, expression of markers for tight and adherens junctions in areas of FITC-albumin leakage consistently remained unaltered in the applied models. However, lectin staining with isolectin B4 indicated structural alterations in the endothelium, which were confirmed by electron microscopy. While ultrastructural alterations in endothelial cells did not differ between the applied models including the reperfusion scenario, we regularly identified vascular alterations, which we propose to reflect four distinct stages of BBB breakdown with ultimate loss of endothelial cells. Therefore, our data strongly suggest that ischemia-related BBB failure is predominantly caused by endothelial degeneration. Thus, protecting endothelial cells may represent a promising therapeutical approach in addition to the established recanalizing strategies.

BACKGROUND: Muller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Muller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Muller cells from the retina of crocodiles remain, hitherto, unknown. METHODS: We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus), endowed with both diurnal and nocturnal vision, by (i) immunohistochemistry, (ii) whole-cell voltage-clamp, and (iii) fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications. RESULTS: Immunohistochemistry revealed that caiman Muller cells, similarly to other vertebrates, express vimentin, GFAP, S100beta, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Muller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Muller cells. Electrophysiological properties of enzymatically dissociated Muller cells without photoreceptors and isolated Muller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Muller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Muller cells thus revealing a uni-directional dye coupling. CONCLUSION: Our data indicate that caiman Muller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K+ buffering.

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β-methylene adenosine 5′-triphosphate; α,β-meATP), P2Y1,12 (adenosine 5′-O-(2-thiodiphosphate; ADP-β-S) and P2Y2,4,6 receptors (uridine 5′-O-(3-thiotriphosphate; UTP-γ-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2′-deoxyuridine (BrdU). The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-β-S and α,β-meATP, whereas UTP-γ-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of α,β-meATP or ADP-β-S. In contrast, UTP-γ-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes. British Journal of Pharmacology (2001) 134, 1180–1189; doi:10.1038/sj.bjp.0704353

Purpose: To determine the visco-elastic properties of isolated rabbit scleral tissue and dose-dependent biomechanical and morphological changes after collagen cross-linking by riboflavin/blue light treatment. Material: Scleral patches from 87 adult albino rabbit eyes were examined by dynamic shear rheology. Scleral patches were treated by riboflavin and different intensities of blue light (450 nm), and the impact on the visco-elastic properties was determined by various rheological test regimes. The relative elastic modulus was calculated from non-treated and corresponding treated scleral patches, and treatments with different blue light intensities were compared. Results: Shear rheology enables us to study the material properties of scleral tissue within physiological relevant parameters. Cross-linking treatment increased the viscous as well as the elastic modulus and changed the ratio of the elastic versus viscous proportion in scleral tissue. Constant riboflavin application combined with different blue light intensities from 12 mW/cm 2 up to 100 mW/cm 2 increased the relative elastic modulus of scleral tissue by factors up to 1.8. Further enhancement of the applied light intensity caused a decline of the relative elastic modulus. This might be due to destructive changes of the collagen bundle structure at larger light intensities, as observed by histological examination. Conclusion: Collagen cross-linking by riboflavin/blue light application increases the biomechanical stiffness of the sclera in a dose-dependent manner up to certain light intensities. Therefore, this treatment might be a suitable therapeutic approach to stabilize the biomechanical properties of scleral tissue in cases of pathological eye expansion.

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na+ and Ca2+currents as well as delayed-rectifier K+ currents. Immunohistochemical staining confirmed that most neurons have Na+ channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K+ inward currents and depolarization-induced K+ outward currents. Glia showed large “passive” currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.

Background/Aims: Osmotic swelling of Müller cells is a common phenomenon in animal models of ischemic and diabetic retinopathies. Müller cells possess a swelling-inhibitory purinergic signaling cascade which can be activated by various receptor ligands including vascular endothelial growth factor (VEGF) and glutamate. Here, we investigated whether deletion of P2Y1 (P2Y1R) and adenosine A1 receptors (A1AR), and of inositol-1,4,5-trisphosphate-receptor type 2 (IP3R2), in mice affects the inhibitory action of VEGF and glutamate on Müller cell swelling. Methods: The cross-sectional area of Müller cell somata was recorded after a 4-min superfusion of retinal slices with a hypoosmotic solution. Results: Hypoosmolarity induced a swelling of Müller cells from P2Y1R-/-, A1AR-/- and IP3R2-/- mice, but not from wild-type mice. Swelling of wild-type Müller cells was induced by hypoosmotic solution containing barium chloride. Whereas VEGF inhibited the swelling of wild-type Müller cells, it had no swelling-inhibitory effect in cells from A1AR-/- and IP3R2-/- mice. Glutamate inhibited the swelling of wild-type Müller cells but not of cells from P2Y1R-/-, A1AR-/- and IP3R2-/- animals. Conclusion: The swelling-inhibitory effects of VEGF and glutamate in murine Müller cells is mediated by transactivation of P2Y1R and A1AR, as well as by intracellular calcium signaling via activation of IP3R2., OA-Komponente aus Allianzlizenz