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1.
Exp Eye Res ; 173: 160-178, 2018 08.
Article in English | MEDLINE | ID: mdl-29753728

ABSTRACT

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.


Subject(s)
Adaptation, Ocular/physiology , Alligators and Crocodiles , Night Vision/physiology , Retina/ultrastructure , Animals , Cell Count , Female , Male , Microscopy, Electron , Photoreceptor Cells, Vertebrate/ultrastructure , Retina/physiology , Retinal Pigment Epithelium/ultrastructure
2.
Exp Eye Res ; 173: 91-108, 2018 08.
Article in English | MEDLINE | ID: mdl-29763583

ABSTRACT

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.


Subject(s)
Alligators and Crocodiles/physiology , Ependymoglial Cells/physiology , Light , Night Vision/physiology , Retina/physiology , Vision, Ocular/physiology , Animals , Eye Proteins/metabolism , Female , Male , Microscopy, Confocal , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology
3.
Proc Biol Sci ; 284(1859)2017 Jul 26.
Article in English | MEDLINE | ID: mdl-28724733

ABSTRACT

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.


Subject(s)
Biological Evolution , Central Nervous System/cytology , Ependymoglial Cells/cytology , Neuroglia/cytology , Animals , Neurons
4.
Graefes Arch Clin Exp Ophthalmol ; 254(8): 1567-1577, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27270346

ABSTRACT

PURPOSE: We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes. METHODS: The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5 %) followed by irradiation with blue light (200, 400, 650 mW/cm(2)) for 20 min. After 3 weeks, the ultrastructure of scleral cells and the abundance of small- (10-100 nm) and large-diameter (>100 nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy. RESULTS: In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200 mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma. CONCLUSIONS: The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200 mW/cm(2) for 20 min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400 mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.


Subject(s)
Collagen/pharmacology , Cross-Linking Reagents/pharmacology , Light , Myopia/therapy , Riboflavin/pharmacology , Sclera/ultrastructure , Animals , Biomechanical Phenomena , Disease Models, Animal , Microscopy, Electron , Myopia/physiopathology , Photosensitizing Agents/pharmacology , Rabbits , Sclera/drug effects , Sclera/physiopathology
5.
Graefes Arch Clin Exp Ophthalmol ; 254(1): 109-22, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26597112

ABSTRACT

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.


Subject(s)
Cross-Linking Reagents , Eye/growth & development , Photochemotherapy , Photosensitizing Agents/pharmacology , Riboflavin/pharmacology , Sclera/drug effects , Sclera/metabolism , Animals , Axial Length, Eye/drug effects , Collagen/metabolism , Eye/diagnostic imaging , Immunohistochemistry , Light , Magnetic Resonance Imaging , Rabbits , Sensory Thresholds , Ultrasonography
6.
Exp Eye Res ; 139: 37-47, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26208440

ABSTRACT

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm(2) (15 to 650 mW/cm(2) for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm(2) (400 mW/cm(2)) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range.


Subject(s)
Cross-Linking Reagents/pharmacology , Riboflavin/pharmacology , Sclera/drug effects , Animals , Biomechanical Phenomena , Disease Models, Animal , Light , Microscopy, Electron , Photosensitizing Agents/pharmacology , Rabbits , Sclera/radiation effects , Sclera/ultrastructure
7.
Acta Ophthalmol ; 93(5): e328-e336, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25516112

ABSTRACT

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.


Subject(s)
Collagen/metabolism , Cross-Linking Reagents , Elastic Modulus/physiology , Light , Riboflavin/pharmacology , Sclera/drug effects , Sclera/metabolism , Animals , Biomechanical Phenomena , Dose-Response Relationship, Radiation , Photochemotherapy , Photosensitizing Agents/pharmacology , Rabbits , Rheology
8.
PLoS One ; 9(5): e97155, 2014.
Article in English | MEDLINE | ID: mdl-24831221

ABSTRACT

BACKGROUND: Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller 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 Müller 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 Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100ß, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling. CONCLUSION: Our data indicate that caiman Müller 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.


Subject(s)
Ependymoglial Cells/cytology , Photoreceptor Cells, Vertebrate/cytology , Potassium Channels, Inwardly Rectifying/metabolism , Retina/physiology , Alligators and Crocodiles/metabolism , Animals , Fluorescent Dyes/chemistry , Glutamates/metabolism , Ion Channel Gating , Membrane Potentials , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Potassium/chemistry , Potassium Channels, Tandem Pore Domain/metabolism , Protein Structure, Tertiary , Retina/metabolism , Signal Transduction
9.
Prog Retin Eye Res ; 38: 43-69, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24157316

ABSTRACT

This article presents a summary and critical review of what is known about the 'grouped retina', a peculiar type of retinal organization in fish in which groups of photoreceptor cell inner and outer segments are arranged in spatially separated bundles. In most but not all cases, these bundles are embedded in light-reflective cups that are formed by the retinal pigment epithelial cells. These cups constitute a specialized type of retinal tapetum (i.e., they are biological 'mirrors' that cause eye shine) and appear to be optimized for different purposes in different fishes. Generally, the large retinal pigment epithelial cells are filled with light-reflecting photonic crystals that consist of guanine, uric acid, or pteridine depending on species, and which ensure that the incoming light becomes directed onto the photoreceptor outer segments. This structural specialization has so far been found in representatives of 17 fish families; of note, not all members of a given family must possess a grouped retina, and the 17 families are not all closely related to each other. In many cases (e.g., in Osteoglossomorpha and Aulopiformes) the inner surface of the cup is formed by three to four layers of strikingly regularly shaped and spaced guanine platelets acting as an optical multilayer. It has been estimated that this provides an up to 10fold increase of the incident light intensity. In certain deep-sea fish (many Aulopiformes and the Polymixidae), small groups of rods are embedded in such 'parabolic mirrors'; most likely, this is an adaptation to the extremely low light intensities available in their habitat. Some of these fishes additionally possess similar tapetal cups that surround individual cones and, very likely, also serve as amplifiers of the weak incident light. In the Osteoglossomorpha, however, that inhabit the turbid water of rivers or streams, the structure of the cups is more complex and undergoes adaptation-dependent changes. At dim daylight, probably representing the usual environmental conditions of the fish, the outer segments of up to 30 cone cells are placed at the bottom of the cup where light intensity is maximized. Strikingly, however, a large number of rod receptor cells are positioned behind each mirroring cup. This peculiar arrangement (i) allows vision at deep red wavelenghts, (ii) matches the sensitivity of rod and cone photoreceptors, and (iii) facilitates the detection of low-contrast and color-mixed stimuli, within the dim, turbid habitat. Thus, for these fish the grouped retina appears to aid in reliable and quick detection of large, fast moving, biologically relevant stimuli such as predators. Overall, the grouped retina appears as a peculiar type of general retinal specialization in a variety of fish species that is adaptive in particular habitats such as turbid freshwater but also the deep-sea. The authors were prompted to write this review by working on the retina of Gnathonemus petersii; the data resulting from this work (Landsberger et al., 2008; Kreying et al., 2012) are included in the present review.


Subject(s)
Fishes/anatomy & histology , Mesopic Vision/physiology , Photoreceptor Cells, Vertebrate/physiology , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/physiology , Visual Perception/physiology , Animals , Light
10.
Invest Ophthalmol Vis Sci ; 53(7): 4170-6, 2012 Jun 28.
Article in English | MEDLINE | ID: mdl-22669714

ABSTRACT

PURPOSE: To study physiologic characteristics of human Müller cells from healthy and pathologically altered eyes. METHODS: Human tissue was used from organ donors and from patients affected with uveal melanoma. Several melanoma eyes also showed retinal detachment. Incubation of freshly prepared slices with a commercial vital dye preferentially stained Müller cells. The Müller cell response to hypotonic stress was observed by recording the cross-sectional area of cell somata. Electrophysiologic properties were investigated in parallel in whole-cell patch-clamp experiments. RESULTS: Inward K+ currents mediated by inwardly rectifying Kir channels were significantly decreased in Müller cells from eyes with uveal melanoma compared with healthy controls. This was accompanied by a decrease of the membrane potential. Both effects were stronger in cells from eyes where the melanoma had caused a widespread retinal detachment. Application of a hypotonic solution did not affect Müller cells from healthy organ donors. By contrast, Müller cells from some melanoma eyes increased their soma size in response to hypotonic solution. This effect was aggravated in cells from eyes with widespread retinal detachment. The inflammatory mediator, arachidonic acid, could induce Müller cell swelling, whereas anti-inflammatory substances reduced the swelling. CONCLUSIONS: The experiments with human tissue confirm earlier data from animal models for retinal pathologies about typical alterations of reactive Müller cells. Hypotonic stress induced Müller cell swelling preferentially in cells from melanoma-affected eyes that displayed decreased inward current amplitudes. Widespread melanoma-associated retinal detachment potentiated the pathologic alterations of Müller cells.


Subject(s)
Melanoma/physiopathology , Neuroglia/physiology , Retinal Ganglion Cells/physiology , Uveal Neoplasms/physiopathology , Humans , Immunohistochemistry , Melanoma/pathology , Membrane Potentials/physiology , Neuroglia/cytology , Patch-Clamp Techniques , Tumor Cells, Cultured , Uveal Neoplasms/pathology
11.
J Neurosci Res ; 89(12): 2018-27, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21538466

ABSTRACT

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.


Subject(s)
Astrocytes/metabolism , Hepatic Encephalopathy/metabolism , Liver Failure/metabolism , Potassium Channels, Inwardly Rectifying/biosynthesis , Animals , Astrocytes/drug effects , Astrocytes/pathology , Blotting, Western , Cells, Cultured , Cerebral Cortex/metabolism , Down-Regulation , Excitatory Amino Acid Transporter 2/biosynthesis , Glutamine/pharmacology , Hepatic Encephalopathy/pathology , Male , Rats , Rats, Long-Evans , Rats, Wistar , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sodium-Potassium-Chloride Symporters/biosynthesis , Solute Carrier Family 12, Member 2
12.
Glia ; 59(2): 256-66, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21154559

ABSTRACT

High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration-dependent, with half-maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative-nitrosative stress, and (iv) inhibitors of phospholipase A(2) and cyclooxygenase. Glutamine-induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A(1) receptors. The data suggest that hypoosmolarity accelerates the swelling-inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative-nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction.


Subject(s)
Cell Size/drug effects , Glutamine/pharmacology , Neuroglia/cytology , Neuroglia/drug effects , Osmosis , Retina/cytology , Adenosine/pharmacology , Adenosine A1 Receptor Antagonists/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Arachidonic Acid/pharmacology , Diazooxonorleucine/pharmacology , Dinoprostone/pharmacology , Drug Interactions , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Membrane Potential, Mitochondrial/drug effects , Osmosis/drug effects , Rats , Rats, Long-Evans , Retinal Ganglion Cells/drug effects , Xanthines/pharmacology
13.
Exp Eye Res ; 92(1): 87-93, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21111734

ABSTRACT

Osmotic swelling of retinal glial (Müller) cells may contribute to the development of edema in diabetic retinopathy. Here, we tested whether oxidative stress and mitochondrial dysfunction are pathogenic factors involved in the osmotic swelling of Müller cells in retinal slices from control and streptozotocin-injected hyperglycemic rats. Hypotonic challenge did not change the size of Müller cell somata from control animals but induced soma swelling in Müller cells of diabetic animals. Administration of a reducing agent blocked the osmotic swelling of Müller cell somata. In retinal tissues from control animals, administration of the reducing agent blocked also the swelling-inducing effects of antagonists of P2Y1 and adenosine A1 receptors. In tissues from diabetic animals, inhibition of xanthine oxidase decreased the soma swelling by approximately 50% while inhibition of NADPH oxidase and nitric oxide synthase had no effects. Blockade of mitochondrial oxidative stress by perindopril, as well as of mitochondrial permeability transition by cyclosporin A or minocycline, attenuated the swelling. In addition, activation of mitochondrial K(ATP) channels by pinacidil fully prevented the swelling. The data suggest that oxidative stress produced by xanthine oxidase, as well as the mitochondria, are implicated in the induction of osmotic swelling of Müller cells from diabetic rats.


Subject(s)
Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Mitochondrial Diseases/metabolism , Neuroglia/pathology , Oxidative Stress , Retinal Neurons/pathology , Animals , Mitochondrial Diseases/pathology , NADPH Oxidases/antagonists & inhibitors , Nitric Oxide Synthase/antagonists & inhibitors , Purinergic P2Y Receptor Antagonists/pharmacology , Rats , Rats, Long-Evans , Receptor, Adenosine A1/metabolism , Retinal Neurons/drug effects , Xanthine Oxidase/antagonists & inhibitors
14.
Invest Ophthalmol Vis Sci ; 50(5): 2359-67, 2009 May.
Article in English | MEDLINE | ID: mdl-18806298

ABSTRACT

PURPOSE: In a rat model of branch retinal vein occlusion (BRVO), changes in gene expression of factors implicated in the development of retinal edema and alterations in the properties of Müller cells were determined. METHODS: In adult Long-Evans rats, BRVO was induced by laser photocoagulation of retinal veins; untreated eyes served as controls. The mRNA levels of after factors were determined with real-time RT-PCR in the neural retina and retinal pigment epithelium after 1 and 3 days of BRVO: VEGF-A, pigment epithelium-derived factor (PEDF), tissue factor, prothrombin, the potassium channel Kir4.1, and aquaporins 1 and 4. Potassium currents were recorded in isolated Müller cells, and cellular swelling was assessed in retinal slices. RESULTS: In the neural retina, the expression of VEGF was upregulated within 1 day of BRVO and returned to the control level after 3 days. PEDF was upregulated in the neuroretina and retinal pigment epithelium after 3 days of BRVO. Prothrombin, Kir4.1, and both aquaporins were downregulated in the neuroretina. After BRVO, Müller cells displayed a decrease in their potassium currents and an altered distribution of Kir4.1 protein, an increase in the size of their somata, and cellular swelling under hypoosmotic stress that was not observed in control tissues. CONCLUSIONS: BRVO results in a rapid transient increase in the expression of VEGF and a delayed increase in the expression of PEDF. The downregulation of Kir4.1 and aquaporins, the mislocation of Kir4.1 protein, and the osmotic swelling of Müller cells may contribute to the development of edema and neuronal degeneration.


Subject(s)
Eye Proteins/genetics , Gene Expression Regulation/physiology , Retina/metabolism , Retinal Neurons/physiology , Retinal Vein Occlusion/genetics , Animals , Aquaporin 1/genetics , Aquaporin 4/genetics , Disease Models, Animal , Electrophysiology , Fluorescent Antibody Technique, Indirect , Glial Fibrillary Acidic Protein/metabolism , Macular Edema/genetics , Macular Edema/metabolism , Membrane Potentials , Nerve Growth Factors/genetics , Potassium Channels, Inwardly Rectifying/genetics , Prothrombin/genetics , RNA, Messenger/metabolism , Rats , Rats, Long-Evans , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Retinal Vein Occlusion/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Thromboplastin/genetics , Vascular Endothelial Growth Factor A/genetics , Vimentin/metabolism
15.
Int J Dev Neurosci ; 26(7): 745-51, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18672046

ABSTRACT

A decrease in the expression of inwardly rectifying potassium (Kir) currents is a characteristic feature of retinal glial (Müller) cells in various retinopathies, e.g., after transient retinal ischemia. We used short-term retinal organ cultures to investigate whether similar physiological alterations can be induced under in vitro conditions. During 4 days in vitro, Müller cells displayed a decrease in Kir currents and an increase in transient A-type potassium currents which was similar to the alterations in membrane physiology during ischemia-reperfusion in vivo. In addition, gliosis of Müller cells both in vivo and in organ cultures was associated with cellular hypertrophy and an alteration in osmotic swelling characteristics. Whereas Müller cells in control retinae did not swell under hypotonic stress, cells in postischemic retinae and in organ cultures swelled upon hypotonic challenge. Therefore, Müller cells in organ cultures can be used to investigate distinct aspects of ischemia-induced Müller cell gliosis. Both the decrease in Kir currents and the alteration in osmotic swelling may reflect a dysfunction of Müller cells regarding the control of the ionic and osmotic homeostasis in the retina.


Subject(s)
Brain Ischemia/physiopathology , Gliosis/physiopathology , Neuroglia/physiology , Retinal Diseases/physiopathology , Water-Electrolyte Balance/physiology , Animals , Aquaporin 4/metabolism , Biomarkers/metabolism , Brain Ischemia/pathology , Cell Size/drug effects , Cells, Cultured , Glial Fibrillary Acidic Protein/metabolism , Gliosis/pathology , Hypotonic Solutions/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neuroglia/drug effects , Neuroglia/pathology , Organ Culture Techniques , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Channels, Inwardly Rectifying/physiology , Rats , Rats, Long-Evans , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Retina/drug effects , Retina/pathology , Retina/physiopathology , Retinal Diseases/pathology , Water-Electrolyte Balance/drug effects
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