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Blue light (BL)-induced retinal injury has become a very common problem due to over exposure to blue light-emitting sources. This study aimed to investigate the possible ameliorating impact of stromal vascular fraction cells (SVFCs) on BL-induced retinal injury. Forty male albino rats were randomly allocated into four groups. The control group rats were kept in 12-h light/12-h dark. Rats of SVFC-control as the control group, but rats were intravenously injected once by SVFCs. Rats of both the BL-group and BL-SVFC group were exposed to BL for 2 weeks; then rats of the BL-SVFC group were intravenously injected once by SVFCs. Following the BL exposure, rats were kept for 8 weeks. Physical and physiological studies were performed; then retinal tissues were collected for biochemical and histological studies. The BL-group showed physical and physiological changes indicating affection of the visual function. Biochemical marker assessment showed a significant increase in MDA, TLR4 and MYD88 tissue levels with a significant decrease in TAC levels. Histological and ultrastructural assessment showed disruption of the normal histological architecture with retinal pigment epithelium, photoreceptors, and ganglion cell deterioration. A significant increase in NF-κB, caspase-3, and GFAP immunoreactivity was also detected. BL-SVFC group showed a significant improvement in physical, physiological, and biochemical parameters. Retinal tissues revealed amelioration of retinal structural and ultrastructural deterioration and a significant decrease in NF-κB and caspase-3 immunoreactivity with a significant increase in GFAP immunoreaction. This study concluded that SVFCs could ameliorate the BL-induced retinal injury through TLR-4/MYD-88/NF-κB signaling inhibition, regenerative, anti-oxidative, and anti-apoptotic effects., Competing Interests: Declarations. Ethical approval: This study was approved by the Research Ethics Committee (Faculty of Medicine, Minia University, Egypt) (Approval No. 404: 6/2022). Conflicts of interest: There is no declaration of competing interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To observe the morphology and physiology of the retina in rats 11 weeks after a constant (24-hour) but moderate (500-lux) illumination for 1 week., Methods: Levels of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, and taurine were measured by high-pressure liquid chromatography (HPLC) in the retina and vitreous humor of albino (Wistar) and pigmented (Long-Evans) rats. Semithin sections were used to determine retinal morphology. The TUNEL method was used to detect cells degenerating by apoptosis. Because the GABAergic system has been shown to be particularly sensitive to the loss of photoreceptors, an additional immunohistochemical study using anti-GABA, anti-glutamate decarboxylase (GAD)(67) and anti-GAD(65) antibodies was performed., Results: No apparent morphologic changes were found in the retina of pigmented rats after constant illumination, whereas in albino rats disappearance of photoreceptors (except in the extreme retinal periphery) and cell bodies was observed. A significant number of TUNEL-positive nuclei also occurred in the remaining nuclear and ganglion cell layers. However, no change in the distribution of GABA, GAD(67), and GAD(65) immunoreactivities was found in either strain under constant illumination compared with control animals. Constant illumination affected the retinal levels of aspartate, glutamate, glutamine, glycine in both strains, whereas GABA contents did not change and taurine was decreased only in albino rats. A significant increase of vitreal glutamate levels was also found in both strains and of taurine levels only in albino rats., Conclusions: Phototoxicity can provoke durable retinal alterations beyond the period of lighting, suggesting progressive and probably continuous modifications of retinal physiology, even in pigmented animals in which the retina seems morphologically normal.

The development of synthetic enzymes in the GABAergic system (GAD(67) and GAD(65)) of the rat retina was analyzed from birth to the 4th postnatal week by the reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. As previously observed for GABA, immunoreactive GAD(67) profiles are seen clearly in the inner retinal layers at birth. At the end of the 1st week of postnatal life, immunolabeling is detected in amacrine and/or ganglion cells and in horizontal cells. GAD(67) immunoreactivity is transiently expressed in horizontal cells and disappears during the 3rd postnatal week. GAD(65) however does not develop until the 5th postnatal day. Immunolabeling is detected in the processes layering the inner plexiform layer (IPL) before being detected in the amacrine and/or ganglion cell bodies. The appearance of transcripts for GAD coincided with the appearance of the proteins. A transient form of mRNA transcripts of the GAD(67) gene containing an extra exon (ES-exon) is also observed which disappears progressively from birth to the 4th postnatal week. This form synthesizes a truncated, enzymatically inactive protein, which could participate in the regulation of GABA synthesis from glutamate present at high levels during retinogenesis.

Purpose: To investigate whether retinal cell death observed in an avian glaucoma-like disorder occurs by apoptosis and whether an increase in excitotoxic amino acid concentration in the vitreous humor is associated temporally with cell death in the retina., Methods: Presumptive retinal apoptotic nuclei were identified by histochemical detection of DNA fragmentation (by TdT-dUTP terminal nick-end labeling [TUNEL]), and vitreal concentrations of glutamate and several other amino acids were determined by high-pressure liquid chromatography with fluorometric detection in the al mutant quail (Coturnix coturnix japonica) in which a glaucoma-like disorder develops spontaneously., Results: TUNEL-labeled nuclei were located mostly in the ganglion cell layer (GCL) in the retina of mutant quails 3 months after hatching. However, labeled nuclei were also observed in the inner and outer nuclear layers. At 7 months, most TUNEL-positive nuclei were detected in the inner nuclear layer, whereas labeled cells in the GCL were reduced in number. No TUNEL-labeled nuclei were detected in the retina of control quails at any age. Vitreal concentrations of glutamate and aspartate were significantly increased in 1-month-old mutant quails compared with control animals. Concentrations decreased at 3 months, and no significant differences were observed between strains at 7 months., Conclusions: Presumptive apoptotic cell death is detected from 3 months after hatching in mutant quails and is not restricted to retinal ganglion cells. Cell death appears just after a significant increase in excitotoxic amino acid concentrations in the vitreous humor, suggesting a correlation between both events.

A post-embedding immunogold study was carried out to estimate the immunoreactivity to glutamate in retinal terminals, P axon terminals and dendrites containing synaptic vesicles in the superficial layers of the optic tectum of Vipera. Retinal terminals, identified following either intraocular injection of tritiated proline, horseradish peroxidase (HRP) or short-term survivals after retinal ablation, were observed to be highly glutamate-immunoreactive. A detailed quantitative analysis showed that about 50% of glutamate immunoreactivity was localized over the synaptic vesicles, 35.8% over mitochondria and 14.2% over the axoplasmic matrix. The close association of immunoreactivity with the synaptic vesicles could indicate that Vipera retino-tectal terminals may use glutamate as their neurotransmitter. P axon terminals and dendrites containing synaptic vesicles, strongly gamma-aminobutyric (GABA)-immunoreactive, were shown to be also moderately glutamate-immunoreactive, but two to three times less than retinal terminals. Moreover, in P axon terminals, the glutamate immunoreactivity was denser over mitochondria than over synaptic vesicles, possibly reflecting the 'metabolic' pool of glutamate, which serves as a precursor in the formation of GABA.

Gamma aminobutyric acid (GABA) is one of the earliest neuroactive substances appearing in the developing central nervous system. The distribution and the time course of the appearance of GABA-like immunoreactivity in the retina of the turtle Emys orbicularis were investigated from embryonic stage 13 to hatching. The first GABA-like immunoreactive cells were observed at stage 14. These cells were located in both the scleral third of the neuroblastic layer and the inner layers of the retina. They were identified as presumptive immature horizontal cells and amacrine cells, respectively. The observation of numerous labelled fibers in the nerve fiber layer suggests that some of the GABA-like immunoreactive cells in the layers were ganglion cells. The development of GABA-like immunoreactive cells followed a gradient of maturation from central to peripheral retina. At hatching, the central retina appeared nearly morphologically mature. In conclusion, GABA is present before the morphofunctional maturation of the retina and this precocious existence supports the idea of its involvement in a neurotrophic role preceding the establishment of synaptic connections and neurotransmitter function.

This study is the first demonstration of glial fibrillary acidic protein (GFAP)-immunoreactivity in the retina of the lamprey Lampetra fluviatilis. This immunoreactivity is expressed on one hand, in radial processes and somata which belong to Müller cells and, on the other hand, in horizontal fibers in the intermediate plexus between horizontal cells. The tracing of these fibers to Müller cells or horizontal cells is discussed.

Iron is implicated in ocular diseases such as in age-related macular degeneration. Light is also considered as a pathological factor in this disease. Earlier, two studies reported the influence of constant light environment on the pattern of expressions of iron-handling proteins. Here, we aimed to see the influence of light in 12-h light-12-h dark (12L:12D) cycles on the expression of iron-handling proteins in chick retina. Chicks were exposed to 400 lx (control) and 5000 lx (experimental) light at 12L:12D cycles and sacrificed at variable timepoints. Retinal ferrous ion (Fe 2+ ) level, ultrastructural changes, lipid peroxidation level, immunolocalization and expression patterns of iron-handling proteins were analysed after light exposure. Both total Fe 2+ level (p = 0.0004) and lipid peroxidation (p = 0.002) significantly increased at 12-, 48- and 168-h timepoint (for Fe 2+ ) and 48- and 168-h timepoint (for lipid peroxidation), and there were degenerative retinal changes after 168 h of light exposure. Intense light exposure led to an increase in the levels of transferrin and transferrin receptor-1 (at 168-h) and ferroportin-1, whereas the levels of ferritins, hephaestin, (at 24-, 48- and 168-h timepoint) and ceruloplasmin (at 168-h timepoint) were decreased. These changes in iron-handling proteins after light exposure are likely due to a disturbance in the iron storage pool evident from decreased ferritin levels, which would result in increased intracellular Fe 2+ levels. To counteract this, Fe 2+ is released into the extracellular space, an observation supported by increased expression of ferroportin-1. Ceruloplasmin was able to convert Fe 2+ into Fe 3+ until 48 h of light exposure, but its decreased expression with time (at 168-h timepoint) resulted in increased extracellular Fe 2+ that might have caused oxidative stress and retinal cell damage., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to., (© 2020 American Society for Photobiology.)

This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites (p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity (p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats. [ABSTRACT FROM AUTHOR]

Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors. [ABSTRACT FROM AUTHOR]

All superclasses of retinal neurons, including bipolar cells (BCs), amacrine cells (ACs) and ganglion cells (GCs), display gap junctional coupling. However, coupling varies extensively by class. Heterocellular AC coupling is common in many mammalian GC classes. Yet, the topology and functions of coupling networks remains largely undefined. GCs are the least frequent superclass in the inner plexiform layer and the gap junctions mediating GC-to-AC coupling (GC:AC) are sparsely arrayed amidst large cohorts of homocellular AC:AC, BC:BC, GC:GC and heterocellular AC:BC gap junctions. Here, we report quantitative coupling for identified GCs in retinal connectome 1 (RC1), a high resolution (2 nm) transmission electron microscopy-based volume of rabbit retina. These reveal that most GC gap junctions in RC1 are suboptical. GC classes lack direct cross-class homocellular coupling with other GCs, despite opportunities via direct membrane contact, while OFF alpha GCs and transient ON directionally selective (DS) GCs are strongly coupled to distinct AC cohorts. Integrated small molecule immunocytochemistry identifies these as GABAergic ACs (γ+ ACs). Multi-hop synaptic queries of RC1 connectome further profile these coupled γ+ ACs. Notably, OFF alpha GCs couple to OFF γ+ ACs and transient ON DS GCs couple to ON γ+ ACs, including a large interstitial amacrine cell, revealing matched ON/OFF photic drive polarities within coupled networks. Furthermore, BC input to these γ+ ACs is tightly matched to the GCs with which they couple. Evaluation of the coupled versus inhibitory targets of the γ+ ACs reveals that in both ON and OFF coupled GC networks these ACs are presynaptic to GC classes that are different than the classes with which they couple. These heterocellular coupling patterns provide a potential mechanism for an excited GC to indirectly inhibit nearby GCs of different classes. Similarly, coupled γ+ ACs engaged in feedback networks can leverage the additional gain of BC synapses in shaping the signaling of downstream targets based on their own selective coupling with GCs. A consequence of coupling is intercellular fluxes of small molecules. GC:AC coupling involves primarily γ+ cells, likely resulting in GABA diffusion into GCs. Surveying GABA signatures in the GC layer across diverse species suggests the majority of vertebrate retinas engage in GC:γ+ AC coupling. [ABSTRACT FROM AUTHOR]

Light causes damage to the retina (phototoxicity) and decreases photoreceptor responses to light. The most harmful component of visible light is the blue wavelength (400–500 nm). Different filters have been tested, but so far all of them allow passing a lot of this wavelength (70%). The aim of this work has been to prove that a filter that removes 94% of the blue component may protect the function and morphology of the retina significantly. Three experimental groups were designed. The first group was unexposed to light, the second one was exposed and the third one was exposed and protected by a blue-blocking filter. Light damage was induced in young albino mice (p30) by exposing them to white light of high intensity (5,000 lux) continuously for 7 days. Short wavelength light filters were used for light protection. The blue component was removed (94%) from the light source by our filter. Electroretinographical recordings were performed before and after light damage. Changes in retinal structure were studied using immunohistochemistry, and TUNEL labeling. Also, cells in the outer nuclear layer were counted and compared among the three different groups. Functional visual responses were significantly more conserved in protected animals (with the blue-blocking filter) than in unprotected animals. Also, retinal structure was better kept and photoreceptor survival was greater in protected animals, these differences were significant in central areas of the retina. Still, functional and morphological responses were significantly lower in protected than in unexposed groups. In conclusion, this blue-blocking filter decreases significantly photoreceptor damage after exposure to high intensity light. Actually, our eyes are exposed for a very long time to high levels of blue light (screens, artificial light LED, neons…). The potential damage caused by blue light can be palliated. [ABSTRACT FROM AUTHOR]

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The retina is the first stage of visual processing. It encodes elemental features of visual scenes. Distinct cone bipolar cells provide the substrate for this to occur. They encode visual information, such as color and luminance, a principle known as parallel processing. Few studies have directly examined whether different forms of spatial information are processed in parallel among cone bipolar cells. To address this issue, we examined the spatial information encoded by mouse ON cone bipolar cells, the subpopulation excited by increments in illumination. Two types of spatial processing were identified. We found that ON cone bipolar cells with axons ramifying in the central inner plexiform layer were tuned to preferentially encode small stimuli. By contrast, ON cone bipolar cells with axons ramifying in the proximal inner plexiform layer, nearest the ganglion cell layer, were tuned to encode both small and large stimuli. This dichotomy in spatial tuning is attributable to amacrine cells providing stronger inhibition to central ON cone bipolar cells compared with proximal ON cone bipolar cells. Furthermore, background illumination altered this difference in spatial tuning. It became less pronounced in bright light, as amacrine celldriven inhibition became pervasive among all ON cone bipolar cells. These results suggest that differential amacrine cell input determined the distinct spatial encoding properties among ON cone bipolar cells. These findings enhance the known parallel processing capacity of the retina. [ABSTRACT FROM AUTHOR]

The human retina is constantly affected by light of varying intensity, this being especially true for photoreceptor cells and retinal pigment epithelium. Traditionally, photoinduced damages of the retina are induced by visible light of high intensity in albino rats using the LIRD (light-induced retinal degeneration) model. This model allows study of pathological processes in the retina and the search for retinoprotectors preventing retinal photodamage. In addition, the etiology and mechanisms of retina damage in the LIRD model have much in common with the mechanisms of the development of age-related retinal disorders, in particular, with age-related macular degeneration (AMD). We have studied preventive and therapeutic effects of Visomitin eye drops (based on the mitochondria-targeted antioxidant SkQ1) on albino rat retinas damaged by bright light. In the first series of experiments, rats receiving Visomitin for two weeks prior to illumination demonstrated significantly less expressed atrophic and degenerative changes in the retina compared to animals receiving similar drops with no SkQ1. In the second series, the illuminated rats were treated for two weeks with Visomitin or similar drops without SkQ1. The damaged retinas of the experimental animals were repaired much more effectively than those of the control animals. Therefore, we conclude that Visomitin SkQ1-containing eye drops have pronounced preventive and therapeutic effects on the photodamaged retina and might be recommended as a photoprotector and a pharmaceutical preparation for the treatment of AMD in combination with conventional medicines. [ABSTRACT FROM AUTHOR]

Müller cells, the principal glial cells of the retina, support the synaptic activity by the uptake and metabolization of extracellular neurotransmitters. Müller cells express uptake and exchange systems for various neurotransmitters including glutamate and γ-aminobutyric acid (GABA). Müller cells remove the bulk of extracellular glutamate in the inner retina and contribute to the glutamate clearance around photoreceptor terminals. By the uptake of glutamate, Müller cells are involved in the shaping and termination of the synaptic activity, particularly in the inner retina. Reactive Müller cells are neuroprotective, e.g., by the clearance of excess extracellular glutamate, but may also contribute to neuronal degeneration by a malfunctioning or even reversal of glial glutamate transporters, or by a downregulation of the key enzyme, glutamine synthetase. This review summarizes the present knowledge about the role of Müller cells in the clearance and metabolization of extracellular glutamate and GABA. Some major pathways of GABA and glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. [ABSTRACT FROM AUTHOR]

We have previously reported that β-adrenoceptor agonists dilate retinal blood vessels, but their effects on retinal neurons have been unclear. In this study, we examined the action of the β-adrenoceptor agonist CL316243 against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats. CL316243 was injected into the vitreous cavity before, with, or after intravitreal NMDA injection. Seven days after NMDA injection, cell loss in the ganglion cell layer (GCL) and thinning of the inner plexiform layer were observed. The reduction in the number of cells in the GCL was diminished by injection of CL316243 at 15, 30, 60, or 120 min after NMDA injection, whereas no significant protective effect was observed when CL316243 was administered 240 min after NMDA injection. Neither preinjection of CL316243 30 min before NMDA nor simultaneous injection of CL316243 with NMDA exerted any protective effect. The β-adrenoceptor antagonist L748337 almost completely abolished the protection conferred by CL316243 injection 120 min after NMDA injection. The number of parvalbumin-positive amacrine cells was decreased in eyes examined 1 day after NMDA treatment, but this was prevented by CL316243 injection at 120 min after NMDA injection. These results suggest that CL316243 exerts protective effects against NMDA-induced damage by stimulation of β-adrenoceptors. β-adrenoceptor agonists may be effective candidates for the treatment of retinal diseases associated with glutamate-induced excitotoxicity, including glaucoma and diabetic retinopathy. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 122, 392-403. Abstract Light-induced damage is a widely used model to study retinal degeneration. We examined whether bacterial lipopolysaccharide (LPS) protects the retina against light-induced injury. One day before intense light exposure for 24 h, rats were intravitreally injected with LPS in one eye and vehicle in the contralateral eye. At several time points after light exposure, rats were subjected to electroretinography and histological analysis. Bax, Bcl-xL, p-Akt, and p-Stat3 levels were assessed by Western blotting, and retinal thiobarbituric acid reactive substances levels were measured as an index of lipid peroxidation. One group of animals received injections of dexamethasone, aminoguanidine (an inducible NOS inhibitor), 5-hydroxydecanoic acid (a mitochondrial K+/ATP channel blocker), or wortmannin [a phosphoinositide-3-kinase (PI3K) inhibitor] in order to analyze their effect on the protection induced by LPS. LPS afforded significant morphologic and functional protection in eyes exposed to intense light. Light damage induced an increase in mitochondrial Bax/cytoplasmic Bax ratio, and lipid peroxidation which were prevented by LPS. Dexamethasone and wortmannin (but not aminoguanidine or 5-hydroxydecanoic acid) prevented the effect of LPS. Moreover, wortmannin prevented the effect of LPS on p-Akt levels. These results indicate that LPS provides retinal protection against light-induced stress, probably through a PI3K/Akt-dependent mechanism. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the expression in the mouse retina and the function in a retinal cell of cullin5 that is known as a protein that is associated with ubiquitilation. Methods: The retinal sites of expressing cullin5 were determined by immunohistochemistry with a specific antibody for cullin5. Retinal cells were transfected with HA-tagged cullin5 using an adenovirus system, and retinal cells were exposed to L-glutamate with or without an over-expression of cullin5. Results: Cullin5 was expressed in the retina of C57BL/6N mice and the retinal ganglion cell layer (RGL) of retina was strongly immunostained. Further, cullin5 was localized in the retinal ganglion cell (RGC). An over-expression of cullin5 reduced the percentage of retinal cell deaths induced by L-glutamate. Conclusions: The expression of cullin5 on retinal cells and reduction in the percentage of dead retinal cells induced by L-glutamate suggest that cullin5 has neuroprotective properties in retinal cells. [ABSTRACT FROM AUTHOR]

Background: The inhibitory neurotransmitter gamma-amino-butyric acid (GABA) not only modulates excitability in the mature nervous system but also regulates neuronal differentiation and circuit development. Horizontal cells, a subset of interneurons in the outer retina, are transiently GABAergic during the period of cone photoreceptor synaptogenesis. In rodents, both horizontal cells and cone axonal terminals express GABAA receptors. To explore the possibility that transient GABA expression in mouse neonatal horizontal cells influences the structural development of synaptic connectivity in the outer retina, we examined a mutant in which expression of GAD67, the major synthesizing enzyme for GABA, is selectively knocked out in the retina. Results: Immunocytochemistry and electron microscopy revealed that the assembly of triad synapses involving cone axonal pedicles and the dendrites of horizontal and bipolar cells is unaffected in the mutant retina. Moreover, loss of GABA synthesis in the outer retina did not perturb the spatial distributions and cell densities of cones and horizontal cells. However, there were some structural alterations at the cellular level: the average size of horizontal cell dendritic clusters was larger in the mutant, and there was also a small but significant increase in cone photoreceptor pedicle area. Moreover, metabotropic glutamate receptor 6 (mGluR6) receptors on the dendrites of ON bipolar cells occupied a slightly larger proportion of the cone pedicle in the mutant. Conclusions: Together, our analysis shows that transient GABA synthesis in horizontal cells is not critical for synapse assembly and axonal and dendritic lamination in the outer retina. However, pre- and postsynaptic structures are somewhat enlarged in the absence of GABA in the developing outer retina, providing for a modest increase in potential contact area between cone photoreceptors and their targets. These findings differ from previous results in which pharmacological blockade of GABAA receptors in the neonatal rabbit retina caused a reduction in cone numbers and led to a grossly disorganized outer retina. [ABSTRACT FROM AUTHOR]

Retinal ganglion cell axons within the globe are laden with mitochondria that are unprotected from light (400–760 nm) impinging onto the retina. Light can be absorbed by mitochondrial enzymes such as cytochrome and flavin oxidases causing the generation of reactive oxygen species, and we have suggested this may pose a risk to ganglion cell survival if their energy state is compromised, as may be so in glaucoma or in Leber’s Hereditary Optic Neuropathy. Here, we demonstrate that light (400–760 nm) provokes apoptosis in cultured retinal ganglion-5 cells, and that this effect is enhanced in low serum, and attenuated by various antioxidants. Apoptosis is shown to be caspase independent, involving reactive oxygen species generation and the activation of poly(ADP-ribose) polymerase-1 and apoptosis-inducing factor. We further show that light-induced apoptosis requires the participation of the mitochondrial respiratory chain. This was demonstrated by culturing fibroblasts (BJhTERT cells) in ethidium bromide for 40 days to deplete their mitochondrial DNA and perturb their mitochondrial respiratory chain function (BJhTERT rh0 cells). Only BJhTERT cells, with intact mitochondrial respiratory chain function were affected by light insult. Finally, we show that exposure of anaesthetized pigmented rat eye to white, but not red light, causes changes in the expression of certain retinal mRNAs (neurofilament light, Thy-1 and melanopsin) and optic nerve proteins (neurofilament light and tubulin), suggesting that ganglion cell survival is affected. Our findings support the proposal that the interaction of light, particularly the blue component, with intra-axonal ganglion cell mitochondria may be deleterious under certain circumstances, and suggest that reducing the light energy impinging upon the retina might benefit patients with certain optic neuropathies. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of citicoline on upregulated clusterin and retinal damage induced by kainic acid (KA). Methods: KA was injected into the vitreous of rats. Effects of systemic citicoline treatments were estimated by measuring the thickness of the various retinal layers, immunoblotting, and immunohistochemical techniques. Results: One day after KA injection, the immunoreactivity of clusterin increased significantly. In rats treated with KA plus citicoline, clusterin immunoreactivity was markedly reduced compared to KA-treated rats. Western blot analysis showed that clusterin protein levels were increased in KA-treated rats, but decreased in KA plus citicoline-treated rats. Apoptotic cell death was determined by TUNEL method. Citicoline reduced the expression of clusterin, as well as the expression of TUNEL after KA injection in the rat retina. Conclusion: The increased expression of clusterin following KA injection in the rat retina suggests the presence of neurodegenerative events; citicoline may provide neuroprotection against neuronal cell damage. [ABSTRACT FROM AUTHOR]

Kir5.1 is an inwardly rectifying K[sup +] channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD[sub 65]) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K[sup +]-buffering action of Müller cells. [ABSTRACT FROM AUTHOR]

Retinal γ-aminobutyric acid type C (GABAc) receptors consist of ρ subunits. Here we report our results from a competitive PCR and patch-clamp electrophysiology study quantifying ρ subunit message and characterizing GABA[sub c] receptor-mediated currents at different stages of mouse retinal development. Mouse ρ1 message is first detected at postnatal day 6 (P6), increases significantly until P9 and remains at this level through adulthood, whereas mouse ρ2 message does not appear until P9, peaks at P15 and remains at this level through adulthood. There is an approximate twofold excess of ρ1 compared to ρ2 message at most stages of development, which persists in adulthood. Functional GABA[sub c] receptors are detected in acutely dissociated bipolar cells of P9 or older mouse retina. Early in development (P9-10), GABA[sub c] receptors are composed solely of ρ1 subunits, but subsequently contain ρ1 and/ or ρ2 subunits (by P11 and later). These findings are intriguing because the onset and rapid increase in ρ subunit transcription and functional expression match the initiation and active period of bipolar cell differentiation in retinal development as well as the stage of eye opening and initial visual experience in the rodent. The investigation of mouse ρ subunits here forms a basis for future studies on the role of GABA[sub c] receptors in retinal development. [ABSTRACT FROM AUTHOR]

The effects of visible light, from short to long wavelengths, on the retina were investigated functionally and histologically. The left eyes of Sprague–Dawley albino rats (6-weeks old, n = 6 for each wavelength) were exposed to seven narrow-band wavelengths (central wavelengths, 421, 441, 459, 501, 541, 581, and 615 nm) with bandwidths of 16 to 29 nm (half bandwidth, ±8–14.5 nm) using a xenon lamp source with bandpass filters at the retinal radiant exposures of 340 and 680 J/cm2. The right unexposed eyes served as controls. Seven days after exposure, flash electroretinograms (ERGs) were recorded, and the outer nuclear layer (ONL) thickness was measured. Compared to the unexposed eyes, significant reductions in the a- and b-wave ERG amplitudes were seen in eyes exposed to 460-nm or shorter wavelengths of light. The ONL thickness near the optic nerve head also tended to decrease with exposure to shorter wavelengths. The decreased ERG amplitudes and ONL thicknesses were most prominent in eyes exposed to 420-nm light at both radiant exposures. When the wavelengths were the same, the higher the amount of radiant exposure and the stronger the damage. Compared to the unexposed eyes, the a- and b-waves did not decrease significantly in eyes exposed to 500-nm or longer wavelength light. The results indicate that the retinal damage induced by visible light observed in albino rats depends on the wavelength and energy level of the exposed light. [ABSTRACT FROM AUTHOR]

As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development. [ABSTRACT FROM AUTHOR]

Background: Ligand-driven modulation of the mitochondrial translocator protein 18 kDa (TSPO) was recently described to dampen the neuroinflammatory response of microglia in a retinal light damage model resulting in protective effects on photoreceptors. We characterized the effects of the TSPO ligand XBD173 in the postischemic retina focusing on changes in the response pattern of the major glial cell types of the retina-microglia and Müller cells.Methods: Retinal ischemia was induced by increasing the intraocular pressure for 60 min followed by reperfusion of the tissue in mice. On retinal cell types enriched via immunomagnetic separation expression analysis of TSPO, its ligand diazepam-binding inhibitor (DBI) and markers of glial activation were performed at transcript and protein level using RNA sequencing, qRT-PCR, lipid chromatography-mass spectrometry, and immunofluorescent labeling. Data on cell morphology and numbers were assessed in retinal slice and flatmount preparations. The retinal functional integrity was determined by electroretinogram recordings.Results: We demonstrate that TSPO is expressed by Müller cells, microglia, vascular cells, retinal pigment epithelium (RPE) of the healthy and postischemic retina, but only at low levels in retinal neurons. While an alleviated neurodegeneration upon XBD173 treatment was found in postischemic retinae as compared to vehicle controls, this neuroprotective effect of XBD173 is mediated putatively by its action on retinal glia. After transient ischemia, TSPO as a marker of activation was upregulated to similar levels in microglia as compared to their counterparts in healthy retinae irrespective of the treatment regimen. However, less microglia were found in XBD173-treated postischemic retinae at 3 days post-surgery (dps) which displayed a more ramified morphology than in retinae of vehicle-treated mice indicating a dampened microglia activation. Müller cells, the major retinal macroglia, show upregulation of the typical gliosis marker GFAP. Importantly, glutamine synthetase was more stably expressed in Müller glia of XBD173-treated postischemic retinae and homeostatic functions such as cellular volume regulation typically diminished in gliotic Müller cells remained functional.Conclusions: In sum, our data imply that beneficial effects of XBD173 treatment on the postischemic survival of inner retinal neurons were primarily mediated by stabilizing neurosupportive functions of glial cells. [ABSTRACT FROM AUTHOR]

Background: Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli beta-galactosidase or enhanced green fluorescence protein (EGFP) using PEI.Results: Optimal transfection efficiency was observed with 1 microg/ml of plasmid DNA and 5 microg/ml PEI. Expression of beta-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with approximately 9% of the neurons expressing beta-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector.Conclusions: These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures. [ABSTRACT FROM AUTHOR]