Search

Purpose The development of degenerative lamellar macular holes (DLH) is largely unclear. This study was aimed at documenting with spectral-domain optical coherence tomography the tractional development and morphological alterations of DLH. Methods A retrospective case series of 44 eyes of 44 patients is described. Results The development of DLH is preceded for months or years by tractional deformations of the fovea due to the action of contractile epiretinal membranes (ERM) and/or the partially detached posterior hyaloid, or by cystoid macular edema (CME). DLH may develop after a tractional stretching and thickening of the foveal center, from a foveal pseudocyst, after a detachment of the foveola from the retinal pigment epithelium, a disruption of the foveal structure due to CME, and after surgical treatment of tractional lamellar or full-thickness macular holes (FTMH). The foveal configuration of a DLH can be spontaneously reestablished after short transient episodes of CME and a small FTMH. A DLH can evolve to a FTMH by traction of an ERM. Surgical treatment of a DLH may result in an irregular regeneration of the foveal center without photoreceptors. Conclusions Tractional forces play an important role in the development of DLH and in the further evolution to FTMH. It is suggested that a DLH is the result of a retinal wound repair process after a tractional disruption of the Müller cell cone and a degeneration of Henle fibers, to prevent a further increase in the degenerative cavitations.

Alterations of the visual function during life are associated with changes in the morphological parameters of the outer retinal layers of the fovea. We evaluated age- and sex-related variations of the mean thicknesses of the different retinal layers at the central foveola which provides the maximal visual acuity. The vertical expansions of the following structures were measured on spectral-domain optical coherence tomographic images of 2944 healthy eyes of 1990 subjects with ages between 5 and 85 years: the total thickness of the retinal tissue, the thickness of the retinal pigment epithelium, the lengths of photoreceptors (receptor segments), photoreceptor outer segments (POS), and photoreceptor inner segments (PIS), and the thicknesses of the ellipsoid zone (EZ), myoid zone (MZ), external limiting membrane, outer nuclear layer, Henle fiber layer, and the horizontal layer of the Müller cell cone. We found diverse morphologies of the central photoreceptor layer with different thicknesses of the EZ and interdigitation zone lines. The mean total thickness of the retinal tissue at the central foveola showed three periods: it increased between 5 and about 41 years of age, displayed a plateau until about 52 years, and decreased continuously thereafter. Photoreceptors, POS, and PIS displayed their maximal mean lengths between 5 and about 36 years of age; the lengths decreased continuously between 36 and 85 years of age. Whereas the mean thickness of the EZ did not alter across the life span, the mean thickness of MZ displayed three periods: it increased between 5 and about 21 years of age, showed a plateau until about 36 years, and decreased considerably thereafter. Sex differences were observed for five parameters in eyes of subjects aging between 55 and 85 years. We suggest that the MZ thickness reflects the level of the metabolic activity of photoreceptors. The increase in the MZ thickness, likely reflecting increasing metabolic activity of photoreceptors, might contribute to the improvement of visual function in young subjects. The decrease of the MZ thickness in the fovea of elderly might reflect a decrease of the metabolic activity perhaps resulting from mitochondrial dysfunction which is known to occur in photoreceptors of aged eyes.

The human retina contains three types of glial cells: microglia and two types of macroglia, astrocytes and Müller cells. Macroglia provide homeostatic and metabolic support to photoreceptors and neurons required for neuronal activity. The fovea, the site of the sharpest vision which is astrocyte- and microglia-free, contains two populations of Müller glia: cells which form the Müller cell cone in the foveola and z-shaped Müller cells of the foveal walls. Both populations are characterized by morphological and functional differences. Müller cells of the foveola do not support the activity of photoreceptors and neurons, but provide the structural stability of the foveal tissue and improve the light transmission through the tissue to the photoreceptors. This article gives overviews of the glia of the human retina and the structure and function of both Müller cell types in the fovea, and describes the contributions of astrocytes and Müller cells to the ontogenetic development of the fovea.

Tractional deformations of the fovea mainly arise from an anomalous posterior vitreous detachment and contraction of epiretinal membranes, and also occur in eyes with cystoid macular edema or high myopia. Traction to the fovea may cause partial- and full-thickness macular defects. Partial-thickness defects are foveal pseudocysts, macular pseudoholes, and tractional, degenerative, and outer lamellar holes. The morphology of the foveal defects can be partly explained by the shape of Muller cells and the location of tissue layer interfaces of low mechanical stability. Because Muller cells and astrocytes provide the structural scaffold of the fovea, they are active players in mediating tractional alterations of the fovea, in protecting the fovea from such alterations, and in the regeneration of the foveal structure. Tractional and degenerative lamellar holes are characterized by a disruption of the Muller cell cone in the foveola. After detachment or disruption of the cone, Muller cells of the foveal walls support the structural stability of the foveal center. After tractional elevation of the inner layers of the foveal walls, possibly resulting in foveoschisis, Muller cells transmit tractional forces from the inner to the outer retina leading to central photoreceptor layer defects and a detachment of the neuroretina from the retinal pigment epithelium. This mechanism plays a role in the widening of outer lameller and full-thickness macular holes, and contributes to visual impairment in eyes with macular disorders caused by conractile epiretinal membranes. Muller cells of the foveal walls may seal holes in the outer fovea and mediate the regeneration of the fovea after closure of full-thickness holes. The latter is mediated by the formation of temporary glial scars whereas persistent glial scars impede regular foveal regeneration. Further research is required to improve our understanding of the roles of glial cells in the pathogenesis and healing of tractional macular disorders.

AIM: To document with spectral-domain optical coherence tomography the morphological regeneration of the fovea after resolution of cystoid macular edema (CME) without and with internal limiting membrane (ILM) detachment and to discuss the presumed role of the glial scaffold for foveal structure stabilization. METHODS: A retrospective case series of 38 eyes of 35 patients is described. Of these, 17 eyes of 16 patients displayed foveal regeneration after resolution of CME, and 6 eyes of 6 patients displayed CME with ILM detachment. Eleven eyes of 9 patients displayed other kinds of foveal and retinal disorders associated with ILM detachment. RESULTS: The pattern of edematous cyst distribution, with or without a large cyst in the foveola and preferred location of cysts in the inner nuclear layer or Henle fiber layer (HFL), may vary between different eyes with CME or in one eye during different CME episodes. Large cysts in the foveola may be associated with a tractional elevation of the inner foveal layers and the formation of a foveoschisis in the HFL. Edematous cysts are usually not formed in the ganglion cell layer. Eyes with CME and ILM detachment display a schisis between the detached ILM and nerve fiber layer (NFL) which is traversed by Müller cell trunks. ILM detachment was also found in single eyes with myopic traction maculopathy, macular pucker, full-thickness macular holes, outer lamellar holes, and glaucomatous parapapillary retinoschisis, and in 3 eyes with Müller cell sheen dystrophy (MCSD). As observed in eyes with MCSD, cellophane maculopathy, and macular pucker, respectively, fundus light reflections can be caused by different highly reflective membranes or layers: the thickened and tightened ILM which may or may not be detached from the NFL, the NFL, or idiopathic epiretinal membranes. In eyes with short single or multiple CME episodes, the central fovea regenerated either completely, which included the disappearance of irregularities of the photoreceptor layer lines and the reformation of a fovea externa, or with remaining irregularities of the photoreceptor layer lines. CONCLUSION: The examples of a complete regeneration of the foveal morphology after transient CME show that the fovea may withstand even large tractional deformations and has a conspicuous capacity of structural regeneration as long as no cell degeneration occurs. It is suggested that the regenerative capacity depends on the integrity of the threedimensional glial scaffold for foveal structure stabilization composed of Müller cell and astrocyte processes. The glial scaffold may also maintain the retinal structure after loss of most retinal neurons as in late-stage MCSD.

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O2 atmosphere and addition of CoCl2, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O2 induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y2 and adenosine A1 receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y2 (but not A1) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y2 receptor signaling.

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.

Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O2 and addition of CoCl2, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl2-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y1, P2Y2, P2X7) and CoCl2-induced conditions (P2Y2, P2X7). Exogenous VEGF, TGF-β1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl2-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl2-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

Many eyes with macular pucker are characterized by a centripetal displacement of the inner foveal layers which may result in a disappearance of the foveal pit. In this retrospective case series of 90 eyes with macular pucker of 90 patients, we describe using spectral-domain optical coherence tomography different foveal configurations with ectopic inner foveal layers, document the relationship between posterior vitreous detachment (PVD) and idiopathic epiretinal membrane (ERM) formation and spontaneous and postoperative morphological alterations of the fovea, and propose an active role of Müller cells in the development of foveal herniation. We found that ERM were formed during or after partial perifoveal PVD, or after foveal deformations caused by tissue edema. The ERM-mediated centripetal displacement of the inner foveal layers and in various eyes anterior hyaloidal traction caused a disappearance of the foveal pit and an anterior stretching of the foveola with a thickening of the central outer nuclear layer (ONL). After the edges of the thickened inner layers of the foveal walls moved together, continuous centripetal displacement of the inner foveal layers generated a bulge of the fovea towards the vitreous (foveal herniation). Macular pseudoholes with a herniation of the inner foveal layers show that the outer layer of the protruding foveal walls is the outer plexiform layer (OPL). If the ERM covered the foveal walls and parafova, but not the foveola, the inner layers of the foveal walls were not fully centripetally displaced and the foveal pit was present. The visual acuity of eyes with ectopic inner foveal layers was inversely correlated with the thickness of the foveal center. Spontaneous morphological alterations after disappearance of the foveal pit may include the development of cystoid macular edema or additional thickening of the foveal tissue and foveal herniation. The foveal configuration with ectopic inner layers of the foveal walls and a thick central ONL persisted over longer postoperative time periods. The data show that the centripetal displacement of the inner foveal layers in eyes with macular pucker, which results in a disappearance of the foveal pit, may also generate foveal herniation which is suggested to be caused by contraction of Müller cell processes in the OPL. The centripetal displacement of the inner foveal layers and the formation of foveal herniation are suggested to reverse the foveal pit formation during development.

This review summarizes the present knowledge concerning the retinal localization of the nerve growth factor (NGF), its precursor proNGF, and the receptors TrkA and p75NTR in the developing and mature rodent retina. We further discuss the changes in the expression of NGF and the receptors in experimental models of retinal disorders and diseases like inherited retinitis pigmentosa, retinal detachment, glaucoma, and diabetic retinopathy. Since proNGF is now recognized as a bioactive signaling molecule which induces cell death through p75NTR activation, the role of proNGF in the induction of retinal cell loss under neurodegenerative conditions is also highlighted. In addition, we present the evidences for a potential therapeutic intervention with NGF for the treatment of retinal neurodegenerative diseases. Different strategies have been developed and experimentally tested in mice and rats in order to reduce cell loss and Muller cell gliosis, e.g., increasing the availability of endogenous NGF, administration of exogenous NGF, activation of TrkA, and inhibition of p75NTR. Here, we discuss the several lines of evidence supporting a protective effect of NGF on retinal cell loss, with specific emphasis on photoreceptor and retinal ganglion cell degeneration. A better understanding of the mechanisms underlying the effects of NGF and proNGF in the modulation of neurodegeneration and gliosis in the retina will help to develop efficient therapeutic strategies for various retinal diseases.

Elephants are precocial mammals that are relatively mature as newborns and mobile shortly after birth. To determine whether the retina of newborn elephants is capable of supporting the mobility of elephant calves, we compared the retinal structures of 2 newborn elephants (1 African and 1 Asian) and 2 adult animals of both species by immunohistochemical and morphometric methods. For the first time, we present here a comprehensive qualitative and quantitative characterization of the cellular composition of the newborn and the adult retinas of 2 elephant species. We found that the retina of elephants is relatively mature at birth. All retinal layers were well discernible, and various retinal cell types were detected in the newborns, including Müller glial cells (expressing glutamine synthetase and cellular retinal binding protein; CRALBP), cone photoreceptors (expressing S-opsin or M/L-opsin), protein kinase Cα-expressing bipolar cells, tyrosine hydroxylase-, choline acetyltransferase (ChAT)-, calbindin-, and calretinin-expressing amacrine cells, and calbindin-expressing horizontal cells. The retina of newborn elephants contains discrete horizontal cells which coexpress ChAT, calbindin, and calretinin. While the overall structure of the retina is very similar between newborn and adult elephants, various parameters change after birth. The postnatal thickening of the retinal ganglion cell axons and the increase in ganglion cell soma size are explained by the increase in body size after birth, and the decreases in the densities of neuronal and glial cells are explained by the postnatal expansion of the retinal surface area. The expression of glutamine synthetase and CRALBP in the Müller cells of newborn elephants suggests that the cells are already capable of supporting the activities of photoreceptors and neurons. As a peculiarity, the elephant retina contains both normally located and displaced giant ganglion cells, with single cells reaching a diameter of more than 50 µm in adults and therefore being almost in the range of giant retinal ganglion cells found in aquatic mammals. Some of these ganglion cells are displaced into the inner nuclear layer, a unique feature of terrestrial mammals. For the first time, we describe here the occurrence of many bistratified rod bipolar cells in the elephant retina. These bistratified bipolar cells may improve nocturnal contrast perception in elephants given their arrhythmic lifestyle.

Purpose To document with spectral domain optical coherence tomography the formation and spontaneous closure of small full-thickness macular holes and to propose the active role of Muller cells in macular hole closure. Methods A retrospective case series of five patients with spontaneous closure of macular holes is reviewed. In one patient, foveal images were recorded over a period of 18 months. Results In a 66-year-old man, vitreofoveal traction caused a detachment of the inner Muller cell layer of the foveola from the outer nuclear layer (ONL) which was associated with a large pseudocyst and a horizontal gap in the central ONL. The traction caused an elongation and subsequent disruption of the stalk of the Muller cell cone in the foveola. A small full-thickness macular hole developed when a portion of the inner Muller cell layer of the foveola was pulled out. After phacoemulsification and shortly before the subsequent spontaneous closure of the hole, there were rapid increases in the number and size of the cystic cavities in the foveal walls resulting in a narrowing of the hole. The hole closed by bridging the gap in the inner part of the central ONL; a new inner Muller cell layer of the foveola was formed, and the gap of the external limiting membrane (ELM) was closed. The cystic cavities in the foveal walls rapidly disappeared within 2 weeks after the closure of the hole. One to 2.5 months after hole closure, the thickness of the central ONL increased which decreased the distance between the central ELM and retinal pigment epithelium. In three of the four other patients, the hole also closed by bridging the gap in the inner part of the ONL. Conclusion It is suggested that the spontaneous closure of small macular holes and the subsequent reconstruction of the normal foveal structure are mediated by active mechanisms of Muller cells which resemble those involved in ontogenetic foveal development.

Full-thickness macular holes (FTMH) are an important cause of visual deterioration. However, different modes of FTMH formation are less investigated. It is also not clear whether the development of edematous cysts contributes to FTMH formation. In this retrospective case series of 30 eyes of 30 patients, we describe using spectral-domain optical coherence tomography different modes of FTMH formation. Morphological alterations of established FTMH are shown in 5 eyes of 5 patients. We found in 2 of 30 eyes investigated that anterior hyaloidal traction induced a hyperreflectivity of the inner Müller cell layer of the foveola prior to FTMH formation. In 3 eyes, FTMH were caused by anterior hyaloidal traction which produced foveal pseudocysts that developed to an outer lamellar hole (OLH) characterized by a disruption of the central outer retina. The OLH developed to a FTMH by the disruption of the inner layer of the foveola. FTMH formation from an OLH by hyaloidal traction was observed also in further 7 eyes. In 2 eyes, the OLH, which preceded FTMH formation, was generated by a serous retinal detachment. In 3 eyes, anterior hyaloidal traction caused a detachment of the fovea from the retinal pigment epithelium (RPE); the subsequent disruption of the foveola resulted in a FTMH. Six eyes showed the development of a FTMH from a degenerative lamellar hole (DLH). In 5 eyes with macular pucker, FTMH were formed by traction of epiretinal membranes (ERM) or hyaloidal traction. Two eyes showed the development of a FTMH by anterior or tangential hyaloidal traction likely without a formation of an OLH. FTMH formation from an OLH proceeded with or without an enlargement of cystic cavities in the foveal walls. The formation of FTMH from a DLH, after a detachment of the fovea, and in macular pucker eyes was associated with a formation of cystic cavities in the foveal walls. The best-corrected visual acuity (BCVA) of eyes with an OLH or FTMH was inversely correlated to the base and minimum diameters of the holes, and with the height of the foveal walls; the highest correlation coefficients were found between the BCVA and the base diameter. The data show that FTMH may be formed via different modes by hyaloidal traction and/or traction of ERM, or after a serous retinal detachment. It is suggested that, after FTMH formation, the impaired fluid clearance through the RPE after detachment of the central outer retina causes the development of edematous cysts in the foveal walls which enlarges the FTMH. The BCVA of eyes with an OLH or FTMH mainly depends on the size of the central photoreceptor-free area.

Tractional forces or mechanical stimulation are known to induce calcium responses in retinal glial cells. The aim of the study was to determine the characteristics of calcium responses in Muller glial cells of the avascular guinea pig retina induced by focal mechanical stimulation. Freshly isolated retinal wholemounts were loaded with Mitotracker Deep Red (to fill Muller cells) and the calcium-sensitive dye Fluo-4/AM. The inner retinal surface was mechanically stimulated with a micropipette tip for 10 ms. Stimulation induced two different cytosolic calcium responses in Muller cells with different kinetics in dependence on the distance from the stimulation site. Muller cells near the stimulation site displayed an immediate and long-lasting calcium response with high amplitude. This response was mediated by calcium influx from the extracellular space likely triggered by activation of ATP-insensitive P2 receptors. More distant Muller cells displayed, with a delay of 2.4 s, transient calcium responses which propagated laterally in a wave-like fashion. Propagating calcium waves were induced by a calcium-independent release of ATP from Muller cells near the stimulation site, and were mediated by a release of calcium from internal stores triggered by ATP, acting in part at P2Y1 receptors. The data suggest that mechanically stimulated Muller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Muller cells. Propagating calcium waves may be implicated in the spatial regulation of the neuronal activity and homeostatic glial functions, and may transmit gliosis-inducing signals across the retina. Mechanical stimulation of guinea pig Muller cells induces two calcium responses: an immediate response around the stimulation site and propagating calcium waves. Both responses are differentially mediated by activation of purinergic receptors. GLIA 2016 GLIA 2017;65:62-74.

Water accumulation in retinal glial (Muller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Muller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Muller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Muller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Muller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Muller cells.

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD21/703 HA). Age-matched Sprague-Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD21/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y1 or adenosine A1 receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y1 receptor antagonist was mediated by induction of oxidative-nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD21/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD21/703 rats, the gene expression levels of P2Y1 and A1 receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD21/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y1 receptor activation serve to inhibit the induction of oxidative-nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

Mammalian retinal glial (Muller) cells are known to guide light through the inner retina to photoreceptors (Franze et al., 2007; Proc Natl Acad Sci U S A 104:8287-8292). It was shown that Muller cells transmit predominantly red-green and less violet-blue light (Labin et al., 2014; Nat Commun 5:4319). It is not known whether this optical function is reflected in the cone-to-Muller cell ratio. To determine this ratio in the retinas of mammals with different lifestyle, we evaluated the local densities of cones and Muller cells in the retinas of guinea pigs, rabbits, sheep, red deer, roe deer, domestic pigs, and wild boars. Retinal wholemounts were labeled with peanut agglutinin to mark cones and anti-vimentin antibodies to identify Muller cells. Wholemounts of guinea pig and rabbit retinas were also labeled with anti-S-opsin-antibodies. With the exceptions of guinea pig and pig retinas that had cone-to-Muller cell ratios of above one, the local densities of cones and Muller cells in the retinas of the species investigated were roughly equal. Because the proportion of S-cones is usually low (for example, 5.3% of all cones in the dorsal guinea pig retina expressed S-opsin), it is suggested that Muller cells are mainly coupled to M-cones. Exceptions are the ventral peripheries of guinea pig and rabbit retinas which are specialized areas with high S-cone densities. Here, up to 50% of Muller cells may be coupled to S-cones, and 40% of S-cones may be not coupled to Muller cells. Among the species investigated, the density of Muller cells in the central retina was inversely correlated with the axial length of the eyes. It is suggested that (with the exception of specialized S-cone areas) Muller cells support high acuity vision by predominant guidance of red-green light to M-opsin expressing cones.

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O(2) atmosphere and addition of CoCl(2), respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O(2) induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y(2) and adenosine A(1) receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y(2) (but not A(1)) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y(2) receptor signaling.

Age-related macular degeneration (AMD) is a leading cause of legal blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids comprised of human RPE cells constructed a well-differentiated monolayer of RPE with Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between RPE and Bruch's membrane with expression of many drusen-associated proteins such as amyloid ß and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases such as AMD.

Purpose Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity. Methods Hyperosmotic media were made up with the addition of NaCl or sucrose. Gene expression was quantified with real-time reverse transcription (RT)–PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. DNA binding of AP-1 protein was evaluated with electrophoretic mobility shift assay (EMSA). Results High NaCl and the addition of sucrose triggered expression of the c-Fos gene, but not of the c-Jun gene. High NaCl also increased the levels of c-Fos and phosphorylated c-Jun proteins and the level of DNA binding of AP-1. Hypoosmolarity decreased the expression of the c-Fos and c-Jun genes. NaCl-induced expression of the c-Fos gene was in part mediated by NFAT5. Autocrine/paracrine activation of fibroblast growth factor and adenosine A1 receptors is involved in mediating NaCl-induced expression of the c-Fos gene. Pharmacological inhibition of the AP-1 activity decreased the NaCl-induced expression of the HIF-1α, NFAT5, VEGF, PlGF, and TGF-β2 genes, and prevented the NaCl-induced secretion of PlGF but not of VEGF. Conclusions The data indicate that AP-1 is activated in RPE cells in response to extracellular hyperosmolarity and mediates in part via the NaCl-induced expression of VEGF and PlGF, and secretion of PlGF. It is suggested that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in part via activation of AP-1.

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Intravitreal administration of neurotrophins such as brain-derived neurotrophic factor (BDNF) is known to promote survival of retinal neurons. Here, we show that exogenous BDNF inhibits the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices or freshly isolated cells with a hypoosmotic solution containing barium ions. BDNF also inhibited the osmotic swelling of bipolar cell somata in retinal slices, but failed to inhibit the osmotic soma swelling of freshly isolated bipolar cells. The inhibitory effect of BDNF on Müller cell swelling was mediated by activation of tropomyosin-related kinase B (TrkB) and transactivation of fibroblast growth factor receptors. Exogenous basic fibroblast growth factor (bFGF) fully inhibited the osmotic swelling of Müller cell somata while it partially inhibited the osmotic swelling of bipolar cell somata. Isolated Müller cells displayed immunoreactivity of truncated TrkB, but not full-length TrkB. Isolated rod bipolar cells displayed immunoreactivities of both TrkB isoforms. Data suggest that the neuroprotective effect of exogenous BDNF in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. While BDNF directly acts on Müller cells by activation of TrkB, BDNF indirectly acts on bipolar cells by inducing glial release of factors like bFGF that inhibit bipolar cell swelling.

A fovea is a pitted invagination in the inner retinal tissue (fovea interna) that overlies an area of photoreceptors specialized for high acuity vision (fovea externa). Although the shape of the vertebrate fovea varies considerably among the species, there are two basic types. The retina of many predatory fish, reptilians, and birds possess one (or two) convexiclivate fovea(s), while the retina of higher primates contains a concaviclivate fovea. By refraction of the incoming light, the convexiclivate fovea may function as image enlarger, focus indicator, and movement detector. By centrifugal displacement of the inner retinal layers, which increases the transparency of the central foveal tissue (the foveola), the primate fovea interna improves the quality of the image received by the central photoreceptors. In this review, we summarize ‒ with the focus on Muller cells of the human and macaque fovea ‒ data regarding the structure of the primate fovea, discuss various aspects of the optical function of the fovea, and propose a model of foveal development. The "Muller cell cone" of the foveola comprises specialized Muller cells which do not support neuronal activity but may serve optical and structural functions. In addition to the "Muller cell cone", structural stabilization of the foveal morphology may be provided by the 'z-shaped' Muller cells of the fovea walls, via exerting tractional forces onto Henle fibers. The spatial distribution of glial fibrillary acidic protein may suggest that the foveola and the Henle fiber layer are subjects to mechanical stress. During development, the foveal pit is proposed to be formed by a vertical contraction of the centralmost Muller cells. After widening of the foveal pit likely mediated by retracting astrocytes, Henle fibers are formed by horizontal contraction of Muller cell processes in the outer plexiform layer and the centripetal displacement of photoreceptors. A better understanding of the molecular, cellular, and mechanical factors involved in the developmental morphogenesis and the structural stabilization of the fovea may help to explain the (patho-) genesis of foveal hypoplasia and macular holes.

Previous studies on the ultrastructure of the primate foveola suggested the presence of an inverted cone-like structure which is formed by 25-35 specialized Müller cells overlying the area of high photoreceptor density. We investigated the ultrastructure of the Müller cells in the foveola of a human and macaque retina. Sections through the posterior poles of an eye of a 40 years-old human donor and an eye of an adult cynomolgus monkey (Macaca fascicularis) were investigated with transmission electron microscopy. The foveola consisted of an inner layer (thickness, 5.5-12 μm) which mainly contained somata (including nuclei) and inner processes of Müller cells; this layer overlaid the central Henle fibers and outer nuclear layer. The inner layer contained numerous watery cysts and thin lamelliform and tubular Müller cell processes which spread along the inner limiting membrane (ILM). The cytoplasm of the outer Müller cell processes became increasingly dispersed and electron-lucent in the course towards the outer limiting membrane. The ILM of the foveola was formed by a very thin basal lamina (thickness,40 nm) while the basal lamina of the parafovea was thick (0.9-1 μm). The data show that there are various conspicuous features of foveolar Müller cells. The numerous thin Müller cell processes below the ILM may smooth the inner surface of the foveola (to minimize image distortion resulting from varying light refraction angles at an uneven retinal surface), create additional barriers to the vitreous cavity (compensating the thinness of the ILM), and provide mechanical stability to the tissue. The decreasing density of the outer process cytoplasm may support the optical function of the foveola.

Purpose Variants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells. Methods Hyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT–PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA. Results Extracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1β. Conclusions The expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium-containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post-ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75(NTR) , and was prevented by blockers of metabotropic glutamate, P2Y1 , adenosine A1 , and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line-derived neurotrophic factor and transforming growth factor-β1, but not epidermal growth factor and platelet-derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75(NTR) and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling. Cytotoxic cell swelling contributes to retinal neurodegeneration. Nerve growth factor (NGF) inhibits the osmotic swelling of glial cells by acting at TrkA, release of bFGF, and opening of K(+) and Cl(-) channels. The NGF-induced glial release of cytokines like bFGF inhibits the osmotic swelling of bipolar cells, suggesting that the neuroprotective effect of NGF is in part mediated by prevention of cytotoxic cell swelling.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Muller) cells produce TGF-β2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Muller cells and bovine microvascular retinal endothelial cells (BRECs). Muller cell-derived TGF-β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-β receptors, and stimulated TGF-β-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-β2 as well as by the presence of Muller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Muller cell-derived TGF-β2: (i) binding of TGF-β2 to BRECs is mediated by the type-II TGF-β receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Muller cell-derived TGF-β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-β or TGF-β type-II receptor or blocking the activation of Smads partially abrogated the effect of Muller cell-conditioned media on BRECs. Together, our data suggest that Muller cells release TGF-β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-β therein.

Background/Aims: By the release of antiangiogenic factors, Müller glial cells provide an angiostatic environment in the normal and ischemic retina. We determined whether Müller cells produce thrombospondin-1 (TSP-1), a known inhibitor of angiogenesis. Methods: Secretion of TSP-1 by cultured Müller cells was determined with ELISA. Slices of rat retinas and surgically excised retinal membranes of human subjects were immunostained against TSP-1 and the glial marker vimentin. The effects of TSP-1 on the growth of bovine retinal endothelial cells (BRECs) and activation of ERK1/2 were determined with DNA synthesis and migration assays, and Western blotting, respectively. Results: Cultured Müller cells secrete TSP-1 under normoxic and hypoxic (0.2% O2) conditions. Secretion of TSP-1 was increased in hypoxia compared to normoxia. In rat retinal slices, glial, retinal ganglion, and possibly horizontal cells were stained for TSP-1. Retinal glial cells in preretinal membranes from human subjects with nonhypoxic epiretinal gliosis (macular pucker) and proliferative diabetic retinopathy, respectively, were immunopositive for TSP-1. Exogenous TSP-1 reduced the VEGF-induced proliferation and migration of BRECs and decreased the phosphorylation level of ERK1/2 in BRECs. Conclusion: The data suggest that Müller cells are one major source of TSP-1 in the normal and ischemic retina. Glia-derived TSP1 may inhibit angiogenic responses in the ischemic retina.

To investigate the effect of induced arteriolar constriction (AC) on alterations in gene expression of factors implicated in the development of edema in branch retinal vein occlusion (BRVO). In Brown-Norway rats, BRVO was induced by laser photocoagulation of the veins in one half of the retina. AC of the afferent arterioles was performed 30 min later. We then determined the expression of Vegfa, Vegfb, Pedf, Kir4.1, Aqp4, Aqp1, Il1s, and Il6 with real-time polymerase chain reaction (RT-PCR) in the neuroretina and retinal pigment epithelium (RPE) after 1, 3, and 7 days. Immunostaining against GFAP, aquaporin (AQP)-4, and Kir4.1 was performed on days 1 and 3. BRVO resulted in transient upregulation of Vegfa in the neuroretina on day 1. The expressions of Kir4.1, AQP4, and AQP1 were downregulated, and Il1s and Il6 were strongly upregulated, on days 1 and 3. The retinal distribution of GFAP and AQP4 proteins remained unaltered, while the Kir4.1 protein displayed redistribution from polarized to uniform retinal distribution. AC accelerated the restoration of downregulated Kir4.1, Aqp4, and Aqp1 in the RPE, of Kir4.1 in the neuroretina, and of upregulated Il6 in the neuroretina. AC did not influence the gliotic alterations of Muller cells and the redistribution of the Kir4.1 protein. Constriction of the afferent artery in the BRVO region accelerated the restoration of potassium channels and Il6. These alterations may contribute to faster resorption of retinal edema, and may decrease the level of inflammation.