Melanin protects choroidal blood vessels against light toxicity

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.     

L-Dopa and the Albino Riddle: Content of L-Dopa in the Developing Retina of Pigmented and Albino Mice

Background

The absence or deficiency of melanin as in albinos, has detrimental effects on retinal development that include aberrant axonal projections from eye to brain and impaired vision. In pigmented retinal pigment epithelium (RPE), dihydroxyphenalanine (L-Dopa), an intermediate in the synthetic path for melanin, has been hypothesized to regulate the tempo of neurogenesis. The time course of expression of retinal L-Dopa, whether it is harbored exclusively in the RPE, the extent of deficiency in albinos compared to isogenic controls, and whether L-Dopa can be restored if exogenously delivered to the albino have been unknown.

Methodology/Principal Findings

L-Dopa and catecholamines including dopamine extracted from retinas of pigmented (C57BL/6J) and congenic albino (C57BL/6J-tyr^c2j) mice, were measured throughout development beginning at E10.5 and at maturity. L-Dopa, but not dopamine nor any other catecholamine, appears in pigmented retina as soon as tyrosinase is expressed in RPE at E10.5. In pigmented retina, L-Dopa content increases throughout pre- and postnatal development until the end of the first postnatal month after which it declines sharply. This time course reflects the onset and completion of retinal development. L-Dopa is absent from embryonic albino retina and is greatly reduced in postnatal albino retina compared to pigmented retina. Dopamine is undetectable in both albino and pigmented retinas until after the postnatal expression of the neuronal enzyme tyrosine hydroxylase. If provided to pregnant albino mothers, L-Dopa accumulates in the RPE of the fetuses.

Conclusions

L-Dopa in pigmented RPE is most abundant during development after which content declines. This L-Dopa is not converted to dopamine. L-Dopa is absent or at low levels in albino retina and can be restored to the RPE by administration in utero. These findings further implicate L-Dopa as a factor in the RPE that could influence development, and demonstrate that administration of L-Dopa could be a means to rescue developmental abnormalities characteristic of albinos.     

Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine

Retinas of rats, rabbits, chicks and carp possess enzymes, i.e. serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), which convert serotonin (5-HT) to melatonin, NAT activity and melatonin levels, but not HIOMT activity, show distinct circadian rhythms, with peak values occurring during the dark (night) phase of the 12 h light-dark cycle. Exposure of the animals to light at night inhibited the night-stimulated NAT activity. Treatment of rats and rabbits with the dopaminergic agonist, apomorphine, inhibited the retinal NAT activity. Dopamine levels in the rabbit retina showed diurnal variations, with higher contents seen during the light phase of both the 12 h light-dark cycle with lights on between 06:00–18:00, and that with reversed periods of illumination (lights on between 18:00–06:00). Melatonin potently inhibited the electrically-evoked calcium-dependent release of [³H]dopamine from pieces of retina from both albino and pigmented rabbits. Our results indicate that the light-regulated melatonin-generating system does operate in the vertebrate retina. The present data, together with other findings, suggest that in the retina there is an antagonistic interplay between melatonin and dopamine. Thus, melatonin inhibits dopamine synthesis in, and release from, the retinal dopaminergic cells, whilst dopamine inhibits the night (dark)-stimulated melatonin formation by decreasing NAT activity. Since light increases metabolic activity of the retinal dopaminergic cells (it enhances the amine synthesis, levels and release), it seems likely that the retinal dopamine plays a role of a “light” messenger in the inhibition of melatonin synthesis. It is suggested that an interplay between melatonin and dopamine in the retina is responsible for regulation of those retinal events which follow circadian rhythmicity, and/or are dependent on light-dark conditions.     

L-DOPA is an endogenous ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.     

Ectopic expression of tyrosine hydroxylase in the pigmented epithelium rescues the retinal abnormalities and visual function common in albinos in the absence of melanin

Albino mammals have profound retinal abnormalities, including photoreceptor deficits and misrouted hemispheric pathways into the brain, demonstrating that melanin or its precursors are required for normal retinal development. Tyrosinase, the primary enzyme in melanin synthesis commonly mutated in albinism, oxidizes l-tyrosine to l-dopaquinone using l-3,4-dihydroxyphenylalanine (L-DOPA) as an intermediate product. L-DOPA is known to signal cell cycle exit during retinal development and plays an important role in the regulation of retinal development. Here, we have mimicked L-DOPA production by ectopically expressing tyrosine hydroxylase in mouse albino retinal pigment epithelium cells. Tyrosine hydroxylase can only oxidize l-tyrosine to L-DOPA without further progression towards melanin. The resulting transgenic animals remain phenotypically albino, but their visual abnormalities are corrected, with normal photoreceptor numbers and hemispheric pathways and improved visual function, assessed by an increase of spatial acuity. Our results demonstrate definitively that only early melanin precursors, L-DOPA or its metabolic derivatives, are vital in the appropriate development of mammalian retinae. They further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Localization of 2-[125I]Iodomelatonin Binding Sites in Mammalian Retina

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40-60% of the binding was specific, as determined with both the agonist 6-chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2-[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2-[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2-[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.     

Microchemical analysis of retina layers in pigmented and albino rats by Fourier transform infrared microspectroscopy

Fourier transform infrared (FT-IR) microspectroscopy is a powerful technique that can be used to collect infrared spectra from microscopic regions of tissue sections. The infrared spectra are evaluated to chemically characterize the absorbing molecules. This technique can be applied to normal or diseased tissues. In the latter case, FT-IR microspectroscopy can reveal chemical changes that are associated with discrete regions of lesion sites, which can provide insights into the chemical mechanisms of disease processes. In the present study, FT-IR microspectroscopy was used to analyze sections of retina from normal (pigmented) and albino rats. The outer segments of retinas from pigmented animals were found to have unusually strong absorption values for C&z.dbnd6;C-H unsaturation and carbonyl functional groups. Docosahexaenoic acid (DHA), a major constituent of lipids in the outer segments, also had particularly high absorption values for these functional groups, which suggests that it is responsible for those enhanced absorption values. Absorbance values for the unsaturation and carbonyl functional groups were substantially reduced in the outer segments of retinas from albino animals. This finding, together with data from other studies on light-induced oxidative events in the retina, indicates a loss of DHA by a light-induced mechanism in albino animals. The outer nuclear layer had strong absorbance values for H-C-OH and P&z. dbnd6;O functional groups, which is likely due to the sugar phosphate backbone of DNA. The outer and inner plexiform layers were found to contain greater concentrations of CH(2) and C&z.dbnd6;O functional groups than the outer and inner nuclear layers, which is due to the high concentration of synaptic connections in the former layers. In summary, FT-IR microspectroscopy revealed a unique chemical profile in the outer segments compared to other retinal layers, and this profile was altered in albino animals.     

Evidence for an Antiapoptotic Role of Dopamine in Developing Retinal Tissue

Inhibition of protein synthesis leads to apoptosis in the undifferentiated neuroblastic layer of the retina of newborn rats. We have shown previously that an increase in the intracellular concentration of cyclic AMP prevented apoptosis induced in the retinal neuroblastic layer by inhibition of protein synthesis. In this study, we tested the effects of dopamine on retinal apoptosis and related these effects to the intracellular concentration of cyclic AMP. Both dopamine (100 microM) and the D1-like agonists SKF-38393, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (6-Cl-PB), and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (100 microM) blocked apoptosis induced in the neuroblastic layer by the protein synthesis inhibitor anisomycin. The antiapoptotic effects of the D1-like agonists were not reversed by the D1-like antagonist SCH-23390 (5-100 microM). Both dopamine and D1-like agonists induced a five- to sevenfold increase in the intracellular concentration of cyclic AMP in the retina of newborn rats. The concentration of cyclic AMP induced by the D1-like agonists in the presence of 100 microM SCH-23390 was still at least two- to threefold as high as control values, showing that the activation of adenylyl cyclase by D1-like agonists was reversed only partially by the specific antagonist. The isoquinolinesulfonamide H-89 (20 microM), an inhibitor of cyclic AMP-dependent protein kinase, partially prevented the antiapoptotic effect of 6-Cl-PB. The data show that an early effect of dopamine in the developing retina is the control of programmed cell death. The antiapoptotic effect of dopamine is mediated, at least in part, through an atypical D1-like receptor coupled to stimulation of adenylyl cyclase, followed by activation of cyclic AMP-dependent protein kinase.     

Retinal dopamine D1 and D2 receptors: Characterization by binding or pharmacological studies and physiological functions

1. In the retinal inner nuclear layer of the majority of species, a dopaminergic neuronal network has been visualized in either amacrine cells or the so-called interplexiform cells. 2. Binding studies of retinal dopamine receptors have revealed the existence of both D1- as well D2-subtypes. The D1-subtype was characterized by labeled SCH 23390 (Kd ranging from 0.175 to 1.6 nM and Bmax from 16 to 482 fmol/mg protein) and the D2-subtype by labelled spiroperidol (Kd ranging from 0.087 to 1.35 nM and Bmax from 12 to 1500 fmol/mg protein) and more selectively by iodosulpiride (Kd 0.6 nM and Bmax 82 fmol/mg protein) or methylspiperone (Kd 0.14 nM and Bmax 223 fmol/mg protein). 3. Retinal dopamine receptors have been also shown to be positively coupled with adenylate cyclase activity in most species, arguing for the existence of D1-subtype, whereas in some others (lower vertebrates and rats), a negative coupling (D2-subtype) has been also detected in peculiar pharmacological conditions implying various combinations of dopamine or a D2-agonist with a D1-antagonist or a D2-antagonist in the absence or presence of forskolin. 4. A subpopulation of autoreceptors of D2-subtype (probably not coupled to adenylate cyclase) also seems to be involved in the modulation of retinal dopamine synthesis and/or release. 5. Light/darkness conditions can affect the sensitivity of retinal dopamine D1 and/or D2-receptors, as studied in binding or pharmacological experiments (cAMP levels, dopamine synthesis, metabolism and release). 6. Visual function(s) of retinal dopamine receptors were connected with the regulation of electrical activity and communication (through gap junctions) between horizontal cells mediated by D1 and D2 receptor stimulation. Movements of photoreceptor cells and migration of melanin granules in retinal pigment epithelial cells as well as synthesis of melatonin in photoreceptors were on the other hand mediated by the stimulation of D2-receptors. 7. Other physiological functions of dopamine D1-receptors respectively in rabbit and in embryonic avian retina would imply the modulation of acetylcholine release and the inhibition of neuronal growth cones.     

Endothelin receptors in light-induced retinal degeneration

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.     

Role of dopamine receptors in the mechanism of formation of stress-induced stomach lesions

Experiments on rats with the use of different exposures to stress (generalized electrization and "social stress") have demonstrated that stimulation of dopamine receptors localized in the central nervous system is one of the reasons for stress-induced gastric lesions, particularly for massive hemorrhages. Stimulation of peripheral dopamine receptors seems to have a gastroprotective action. As judged from the intensity of the effects of the dopamine agonists, apomorphine and L-DOPA, on stress-induced lesions of the gastric mucosa, stimulation of D2- rather than of D1-dopamine receptors is of greater importance in stress.     

Preventive and therapeutic effects of SkQ1-containing Visomitin eye drops against light-induced retinal degeneration

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.     

An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA

The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.     

Photobiomodulation Mitigates Diabetes-Induced Retinopathy by Direct and Indirect Mechanisms: Evidence from Intervention Studies in Pigmented Mice

ObjectiveDaily application of far-red light from the onset of diabetes mitigated diabetes-induced abnormalities in retinas of albino rats. Here, we test the hypothesis that photobiomodulation (PBM) is effective in diabetic, pigmented mice, even when delayed until weeks after onset of diabetes. Direct and indirect effects of PBM on the retina also were studied.MethodsDiabetes was induced in C57Bl/6J mice using streptozotocin. Some diabetics were exposed to PBM therapy (4 min/day; 670 nm) daily. In one study, mice were diabetic for 4 weeks before initiation of PBM for an additional 10 weeks. Retinal oxidative stress, inflammation, and retinal function were measured. In some mice, heads were covered with a lead shield during PBM to prevent direct illumination of the eye, or animals were treated with an inhibitor of heme oxygenase-1. In a second study, PBM was initiated immediately after onset of diabetes, and administered daily for 2 months. These mice were examined using manganese-enhanced MRI to assess effects of PBM on transretinal calcium channel function in vivo.ResultsPBM intervention improved diabetes-induced changes in superoxide generation, leukostasis, expression of ICAM-1, and visual performance. PBM acted in part remotely from the retina because the beneficial effects were achieved even with the head shielded from the light therapy, and because leukocyte-mediated cytotoxicity of retinal endothelial cells was less in diabetics treated with PBM. SnPP+PBM significantly reduced iNOS expression compared to PBM alone, but significantly exacerbated leukostasis. In study 2, PBM largely mitigated diabetes-induced retinal calcium channel dysfunction in all retinal layers.ConclusionsPBM induces retinal protection against abnormalities induced by diabetes in pigmented animals, and even as an intervention. Beneficial effects on the retina likely are mediated by both direct and indirect mechanisms. PBM is a novel non-pharmacologic treatment strategy to inhibit early changes of diabetic retinopathy.     

The effects of melatonin and L-DOPA on the diurnal rhythms of free amino acids content in the rat retina

The effects of melatonin and dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) intraperitoneal administration on the rhythms of free amino acids content in the retina of rats were studied. The authors found that the levels of those amino acids, which are protein constituents but not neurotransmitters in the rat retina, change diurnally with maximum at 3-6 h after light onset. Diurnal changes of Ala, Arg, Asn, Ile, Met, Ser, Trp, and Val content persisted in the retina of rats maintained at constant darkness. This fact confirms the true circadian nature of these rhythms. Constant lighting abolished diurnal changes of the content of all amino acids with the exception of Trp. Daytime but not nighttime administration of melatonin decreased the levels of Ala, Asn, Gln, Ile, Met, and Ser down to nocturnal values. Diurnal changes of amino acids content vanished in melatonin-injected rats. The effect of melatonin administration disappeared when the protein synthesis was inhibited by cycloheximide. The effect of intraperitoneal administration of L-DOPA on the levels of free amino acids was opposite the effect of melatonin administration. L-DOPA increased nocturnal levels of Gly, Thr, Trp, and Val but had no effect on the daytime amino acids content. As in the case of melatonin administration, significant diurnal changes of amino acid levels disappeared in L-DOPA-injected rats. The authors hypothesize that melatonin and dopamine can serve as zeitgebers-antagonists of amino acids content rhythms in the rat retina.     

The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific for intermediate filament proteins

Rat retina structure was studied between embryonic day 14 and adult with antibodies specific for vimentin, glial fibrillary acidic protein (GFA) and the proteins of the neurofilament triplet. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond, in the developing retina, to ventriculocytes, and in the mature retina to Müller cells. They could not normally be stained with any of the other intermediate filament antibodies employed here. We did find, however, that some older albino rats possessed GFA staining in addition to vimentin in these processes. Since we never saw such staining in the retinae of mature non-albino rats, and the retinae of older albino rats often showed signs of degeneration, we concluded that such GFA expression was most likely pathological. Neurofilament protein-positive processes were first detectable at embryonic day 15 1/2 in the inner regions of the retina, and corresponded to the axons of retinal ganglion cells. Such processes were equivalently displayed with antibodies to 68 K and 145 K protein, but were negative with 200 K protein. Some 68 K and 145 K positive fibers could also be decorated with vimentin antibody at this stage, though at later stages this was not the case. At later development stages more 68 K and 145 K neurofilament positive processes appeared, and after the first post-natal week progressively more of such processes became in addition 200 K positive, so that almost all neurofilament positive fibers in the adult stained for all three proteins. Such fibers, in the mature retina corresponded to 68 K and 145 K positive optic nerve fibers, and the processes of neurones in the inner plexiform layer. All fibers in the mature optic nerve fiber layer, but not all of those in the inner plexiform layer were stainable with 200 K antibodies. At 4 days post-natal we were able to detect 68 K and 145 K protein positive profiles in the outer regions of the developing retina, the prospective outer plexiform layer. Such profiles were always in addition vimentin positive, but negative for 200 K protein. During further development such profiles became ordered into a well defined layer and from about post-natal day 13 all of them began to acquire 200 K protein. They could be identified as the processes of horizontal cells. They continued to express vimentin in addition to the three triplet proteins in the adult, a so far unprecedented situation. We were able to detect neurofilament staining in the mature retina only in the above described regions, the inner and outer nuclear layer and the photoreceptor processes being completely free of staining. GFA was first detected in short processes adjacent to the inner limiting membrane which penetrated the optic nerve fiber layer. Such profiles were first detectable in the eye of the newborn animal, and were invariably identically stainable with vimentin at this age. These profiles could be stained with both vimentin and GFA at all later stages examined, although GFA staining became very much stronger than vimentin staining in some profiles in the adult. The results presented here are discussed in terms of development of the different retinal cell types.     

Optic nerve sectioning does not affect the development of the retina

The development of the retina of the albino rat was studied after sectioning of the optic nerves on the 2nd postnatal day. The 2nd day represents a stage at which the retina shows only the ganglion cell layer clearly delineated from an undifferentiated mass. Section of optic nerves at this stage did not affect the subsequent retinal development. Both control and experimental eyes developed at the same pace. Some minor degrees of 'retardation' e.g. the sizes of outer segments, appeared to deviate in the experimental retinae.     

Cytochemical Alterations in the Rat Retina by LPS Administration

LPS-induced inflammation and changes in protein phosphorylation and the JAK-STAT pathway accompanying glial activation after LPS treatment, were followed by analyzing secreted proinflammatory cytokine levels. The administration of LPS caused tyrosine phosphorylation of STAT3 in retinae and induced glial fibrillary acidic protein. (GFAP) from the nerve fiber layer to the ganglion cell layer. Our results suggest that the LPS-induced activation of the JAK2/STAT3 signaling pathway may play a key role in the induction of astrogliosis. However, no significant increase in vimentin, OX-42 or inducible nitric oxide synthase (iNOS) expressions were observed after LPS administration. Sphingosine kinase catalyzes the conversion of sphingosine to sphingosine-1–phosphate (So-1-P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, it was found that sphingolipid metabolite levels were elevated in the serum and retinae of LPS-injected rats. To further investigate the chronic effect of increased So-1-P in the retina, So-1-P was infused intracerebroventricularly (i.c.v.) into rats using an osmotic minipump at 100 pmol/10 μl h^-1 for 7 days, and was found to increase retinal GFAP expression. These observations suggest that LPS induces the activation of retinal astrocytes via JAK2/STAT3 and that LPS affects So-1-P generation. Our findings also suggest that elevated So-1-P in the retina and/or in serum could induce cytochemical alterations in LPS treated or inflamed retinae.     

Retinal remodeling in inherited photoreceptor degenerations

Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.