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The induction of retinal degeneration by light exposure is widely used to study mechanisms of cell death. The advantage of such light-induced lesions over genetically determined degenerations is that light exposures can be manipulated according to the needs of the experimenter. Bright white light exposure can induce a synchronized burst of apoptosis in photoreceptors in a large retinal area which permits to study cellular and molecular events in a controlled fashion. Blue light of high energy induces a hot spot of high retinal irradiance within very short exposure durations (seconds to minutes) and may help to unravel the initial events after light absorption which may be similar for all damage regimens. These initial events may then induce various molecular signaling pathways and secondary effects such as lipid and protein oxidation, which may be varying in different light damage setups and different strains or species, respectively. Blue light lesions also allow to study cellular responses in a circumscribed retinal area (hot spot) in comparison with the surrounding tissue.Here we describe the methods for short-term exposures (within the hours range) to bright full-spectrum white light and for short exposures (seconds to minutes) to high-energy monochromatic blue or green light.

The induction of retinal degeneration by light exposure is widely used to study mechanisms of cell death. The advantage of such light-induced lesions over genetically determined degenerations is that light exposures can be manipulated according to the needs of the experimenter. Bright white light exposure can induce a synchronized burst of apoptosis in photoreceptors in a large retinal area which permits to study cellular and molecular events in a controlled fashion. Blue light of high energy induces a hot spot of high retinal irradiance within very short exposure durations (seconds to minutes) and may help to unravel the initial events after light absorption which may be similar for all damage regimens. These initial events may then induce various molecular signaling pathways and secondary effects such as lipid and protein oxidation, which may be varying in different light damage setups and different strains or species, respectively. Blue light lesions also allow to study cellular responses in a circumscribed retinal area (hot spot) in comparison with the surrounding tissue.Here we describe the methods for short-term exposures (within the hours range) to bright full-spectrum white light and for short exposures (seconds to minutes) to high-energy monochromatic blue or green light.

While knowledge about standardization of skin protection against ultraviolet radiation (UVR) has progressed over the past few decades, there is no uniform and generally accepted standardized measurement for UV eye protection. The literature provides solid evidence that UV can induce considerable damage to structures of the eye. As well as damaging the eyelids and periorbital skin, chronic UV exposure may also affect the conjunctiva and lens. Clinically, this damage can manifest as skin cancer and premature skin ageing as well as the development of pterygia and premature cortical cataracts. Modern eye protection, used daily, offers the opportunity to prevent these adverse sequelae of lifelong UV exposure. A standardized, reliable and comprehensive label for consumers and professionals is currently lacking. In this review we (i) summarize the existing literature about UV radiation-induced damage to the eye and surrounding skin; (ii) review the recent technological advances in UV protection by means of lenses; (iii) review the definition of the Eye-Sun Protection Factor (E-SPF®), which describes the intrinsic UV protection properties of lenses and lens coating materials based on their capacity to absorb or reflect UV radiation; and (iv) propose a strategy for establishing the biological relevance of the E-SPF. © 2013 John Wiley & Sons A/S.

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

BackgroundOptical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.Methodology/principal findingsWe achieved to adapt a commercial 3(rd) generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.Conclusions/significanceWe found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.

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

RPE65 is a retinal pigment epithelial protein essential for the regeneration of 11-cis-retinal, the chromophore of cone and rod visual pigments. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber's congenital amaurosis to autosomal recessive retinitis pigmentosa. One of the most frequent missense mutations is an amino acid substitution at position 91 (R91W). Affected patients have useful cone vision in the first decade of life, but progressively lose sight during adolescence. We generated R91W knock-in mice to understand the mechanism of retinal degeneration caused by this aberrant Rpe65 variant. We found that in contrast to Rpe65 null mice, low but substantial levels of both RPE65 and 11-cis-retinal were present. Whereas rod function was impaired already in young animals, cone function was less affected. Rhodopsin metabolism and photoreceptor morphology were disturbed, leading to a progressive loss of photoreceptor cells and retinal function. Thus, the consequences of the R91W mutation are clearly distinguishable from an Rpe65 null mutation as evidenced by the production of 11-cis-retinal and rhodopsin as well as by less severe morphological and functional disturbances at early age. Taken together, the pathology in R91W knock-in mice mimics many aspects of the corresponding human blinding disease. Therefore, this mouse mutant provides a valuable animal model to test therapeutic concepts for patients affected by RPE65 missense mutations.

Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65(450Leu) or Rpe65(450Met)) can act as a modulator of light-damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild-type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c-Fos/AP-1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65(450Met) variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65(450Leu) variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c-Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c-Fos.

In the past few years, the essential role of the homeobox gene Pax6for eye development has been demonstrated unambiguously in a variety of species including humans. In humans, Pax6mutations lead to a variety of ocular malformations of the anterior and posterior segment. However, little is known about PAX6 expression in the adult human retina. We have therefore investigated PAX6 levels and localization in the human retina at various ages. Adult human eyes of various ages (17–79 years) were obtained from the Zurich Eye Bank. PAX6 expression levels and patterns were analysed by Western blot analysis of total retinal protein and by immunohistochemistry on paraffin sections, respectively. PAX6 expression in the retina was detected up to 79 years of donor age and was predominantly localized to the ganglion cell layer and the inner part of the inner nuclear layer. PAX6 remains distinctly expressed throughout the lifespan of the human retina suggesting a role for PAX6 in the retina after completion of eye morphogenesis.

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.

The purpose of this study was to determine the QTL that influence acute, light-induced retinal degeneration differences between the BALB/cByJ and 129S1/SvImJ mouse strains. Five- to 6-week-old F(2) progeny of an intercross between the two strains were exposed to 15,000 LUX of white light for 1 h after their pupils were dilated, placed in the dark for 16 h, and kept for 10-12 days in dim cyclic light before retinal rhodopsin was measured spectrophotometrically. This was used as the quantitative trait for retinal degeneration. Neither gender nor pigmentation had a significant influence on the amount of rhodopsin after light exposure in the F(2) progeny. For genetic study, DNAs of the 27-36 F(2) progeny with the highest and 27-36 F(2) with the lowest levels of rhodopsin after light exposure were genotyped with 71 dinucleotide repeat markers spanning the genome. Any marker with a 95% probability of being associated with phenotype was tested in all 289 F(2) progeny. Data were analyzed with Map Manager QTX. Significant QTL were found on mouse Chrs 1 and 4, and suggestive QTL on Chrs 6 and 2. The four QTL together equal an estimated 78% of the total genetic effect, and each of the QTL represents a gene with BALB/c susceptible alleles. The Chr 6 QTL is in the same region as a highly significant age-related retinal degeneration QTL found previously. Identification of these QTL is a first step toward identifying the modifier genes/alleles they represent, and identification of the modifiers may provide important information for human retinal diseases that are accelerated by light exposure.

Age-related macular degeneration (AMD) is a main causes of severe visual impairment in the elderly in industrialized countries. The pathogenesis of this complex diseases is largely unknown, even though clinical characteristics and histopathology are well described. Because several aging changes are identical to those observed in AMD, there appears to exist an unknown switch mechanism from normal ageing to disease. Recent anatomical studies using elegant innovative techniques reveal that there is a 30% rod loss in normal ageing, which is increased in early AMD. Those and other observations by Curcio and co-workers indicate that early rod loss is an important denominator of AMD (Curcio CA. Eye 2001; 15:376). As in retinitis pigmentosa (RP), rods appear to die by apoptosis. Thus it seems mandatory to study the regulation of rod cell death in animal models to unravel possible mechanisms of rod loss in AMD. Our laboratory investigates signal transduction pathways and gene regulation of rod death in our model of light-induced apoptosis. The transcription factor AP1 is essential, whereas other classical pro- and antiapoptotic genes appear to be less important in our model system. Caspase-1 gene expression is distinctly upregulated after light exposure and there are several factors which completely protect against light-induced cell death, such as the anesthetic halothane, dexamethasone and the absence of bleachable rhodopsin during light exposure. A fast rhodopsin regeneration rate increased damage susceptibility. Our data indicate that rhodopsin is essential for the initiation of light-induced rod loss. Following photon absorption, there may be the generation of photochemically active molecules wich then induce the apoptotic death cascade.

Excessive phototransduction signaling is thought to be involved in light-induced and inherited retinal degeneration. Using knockout mice with defects in rhodopsin shut-off and transducin signaling, we show that two different pathways of photoreceptor-cell apoptosis are induced by light. Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1. By contrast, low light induces an apoptotic pathway that requires transducin. We also provide evidence that additional genetic factors regulate sensitivity to light-induced damage. Our use of defined mouse mutants resolves some of the complexity underlying the mechanisms that regulate susceptibility to retinal degeneration.

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

Lack of the AP-1 member c-Fos protects photoreceptors against light-induced apoptosis, a model for retinal degeneration. In mice, light damage increases the activity of the transcription factor AP-1, while pharmacological suppression of AP-1 prevents apoptosis, suggesting the involvement of pro-apoptotic AP-1 target genes. Recently, however, it was shown that photoreceptors expressing Fra-1 in place of c-Fos (Fos (Fosl1/Fosl1) ) are apoptosis competent despite the lack of transactivation domains in Fra-1. Here, we show that morphological features of light-induced apoptosis were indistinguishable in Fos (Fosl1/Fosl1) and wild-type mice. Furthermore, light exposure comparably increased AP-1 activity in both genotypes. Opposite to wild-type mice, Fra-1, but not c-Fos, was detectable in AP-1 complexes of Fos (Fosl1/Fosl1) mice. Importantly, AP-1 responsiveness for glucocorticoid receptor-mediated inhibition was preserved in Fos (Fosl1/Fosl1) mice. Thus, Fra-1 takes over for c-Fos in pro- and anti-apoptotic signal transduction. As Fra-1 lacks transactivation domains, AP-1 may not induce, but rather suppress genes in retinal light damage.

Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium1, account for 10–15% of LCA cases2,3. Whereas previous studies of RPE65 deficiency in both animal models1,4 and patients5,6 attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65−/− mice by generating double– mutant mice with models of pure cone function7 (rhodopsin-deficient mice; Rho−/−) and pure rod function8 (cyclic nucleotide–gated channel α3–deficient mice; Cnga3−/−). The electroretinograms (ERGs) of Rpe65−/− and Rpe65−/−Cnga3−/− mice were almost identical, whereas there was no assessable response in Rpe65−/−Rho−/− mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.

Apoptosis is essential for retinal development but it is also a major mode of cell loss in many human retinal dystrophies. High levels of visible light induce retinal apoptosis in mice and rats. This process is dependent on the induction of the transcription factor AP-1, a dimeric complex composed of c-Fos and c-Jun/JunD phosphoproteins. While c-Fos is essential, JunD is dispensable for light-induced photoreceptor apoptosis. Here we show that N-terminal phosphorylation of c-Jun, the other main partner of c-Fos in induced AP-1 complexes is not required for programmed cell death during retinal development in vivo and is also dispensable for photoreceptor apoptosis induced by the exogenous stimuli "excessive light" and N-nitroso-N-methylurea (MNU). Mice expressing a mutant c-Jun protein (JunAA) that cannot be phosphorylated at its N-terminus are apoptosis competent and their retina is not distinguishable from wild-type mice. Accordingly, Jun kinase, responsible for phosphorylation of wild-type c-Jun protein is at best only marginally induced by the apoptotic stimuli "light" and MNU. Complex composition of light-induced AP-1 complexes is similar in wild-type and JunAA mice. This shows that the mutant c-Jun protein can be part of the DNA binding complex AP-1 and demonstrates that induction of the DNA binding activity of AP-1 after light insult does not depend on N-terminal phosphorylation of c-Jun. Our results suggest that transactivation of target genes by phosphorylated c-jun/AP-1 is not required for MNU- or light-induced apoptosis of photoreceptor cells.

Excessive light can cause retinal degeneration and may be an environmental cofactor accelerating retinal dystrophies and age-related diseases. In rodent models, the light damage susceptibility (LDS) of the retina is determined genetically. In two mouse strains, with different degrees of LDS, a Leu450Met variation in the pigment epithelial protein RPE65 was shown recently to cosegregate with low LDS. Because light damage is rhodopsin-mediated, and RPE65 is essential for the regeneration of rhodopsin in the visual cycle, we analyzed this variation regarding rhodopsin metabolism and LDS in four mouse strains. We found that, in contrast to previous assertions, LDS does not correlate with the maximal retinal content of rhodopsin present after dark adaptation. Instead, LDS correlated positively with the kinetics of rhodopsin regeneration, which determine rhodopsin availability during light exposure. Light damage occurred after absorption of a threshold dose of photons and thus fast regeneration, as observed in those two strains having Leu at position 450 of RPE65, was correlated with the occurrence of photoreceptor apoptosis after short exposure. In contrast, mice with the Leu450Met variation ofRpe65regenerated rhodopsin with slow kinetics and showed an increased resistance to light-induced retinal degeneration. In these mice, RPE65 protein levels were reduced by a post-transcriptional mechanism. F1hybrid mice, carrying one normal and one variantRpe65gene, had intermediate levels of the corresponding protein and showed intermediate rhodopsin regeneration kinetics and an intermediate LDS. Thus, none of the two variants ofRpe65had a dominant effect.

10-20% of individuals over the age of 65 suffer from age-related macular degeneration (AMD), the leading cause of severe visual impairment in humans living in developed countries. The pathogenesis of this complex disease is poorly understood, and no efficient therapy or prevention exists to date. A precondition for AMD appears to be the accumulation of the age pigment lipofuscin in lysosomes of retinal pigment epithelial (RPE) cells. In AMD, these cells seem to die by apoptosis with subsequent death of photoreceptor cells, and light may accelerate the disease process. Intracellular factors leading to cell death are not known. Here we show that the lipophilic cation N-retinyl-N-retinylidene ethanolamine (A2E), a lipofuscin component, induces apoptosis in RPE and other cells at concentrations found in human retina. Apoptosis is accompanied by the appearance of the proapoptotic proteins cytochrome c and apoptosis-inducing factor in the cytoplasm and the nucleus. Biochemical examinations show that A2E specifically targets cytochrome oxidase (COX). With both isolated mitochondria and purified COX, A2E inhibits oxygen consumption synergistically with light. Inhibition is reversed by the addition of cytochrome c or cardiolipin, a negatively charged phospholipid that facilitates the binding of cytochrome c to membranes. Succinate dehydrogenase activity is not altered by A2E. We suggest that A2E can act as a proapoptotic molecule via a mitochondria-related mechanism, possibly through site-specific targeting of this cation to COX. Loss of RPE cell viability through inhibition of mitochondrial function might constitute a pivotal step toward the progressive degeneration of the central retina.

The dimeric transcription factor AP-1 (activator protein-1) is composed of members of the Fos, Jun, and ATF family of proteins (Angel and Karin 1991). AP-1 activity is induced by a vast number of stimuli, including growth factors, cytokines, and UV irradiation, and couples cell surface signals to changes in cellular phenotype by regulating the expression of target genes (Angel and Karin 1991; Karin et al. 1997). Members of the AP-1 complex are critically involved in a multitude of cellular processes such as proliferation, differentiation, cell death, and oncogenic transformation. It has been hypothesized that the ability to regulate such a large number of biological processes is achieved by the formation of various functional dimers with distinct biological properties. Fos proteins (c-Fos, FosB, Fra-1, Fra-2) form stable dimers with Jun proteins (c-Jun, JunB, JunD), whereas Jun proteins can form homodimers and heterodimers with Fos and ATF proteins (Angel and Karin 1991; Karin et al. 1997). c-Fos (encoded by Fos) and Fra-1 (Fos-related antigen-1, encoded by the Fos-like-1 gene, Fosl1) show high homology in their leucine zipper and DNA-binding domains. Consistent with this sequence conservation, DNA binding specificity of Fra-1/c-Jun heterodimers is indistinguishable from c-Fos/c-Jun heterodimers (Cohen et al. 1989). Outside these domains, the proteins show little similarity. Most importantly, Fra-1 lacks transactivation domains and fails to activate transcription when fused to the DNA-binding domain of Gal4. In contrast, c-Fos is a potent activator of transcription (Cohen et al. 1989). Interestingly, Fosl1 is a transcriptional target of c-Fos, and induction of Fosl1 upon growth factor stimulation is delayed compared with Fos (Cohen and Curran 1988; Bergers et al. 1995; Schreiber et al. 1997; Matsuo et al. 2000). This led to the hypothesis that Fra-1 might counteract the function of c-Fos by lowering the transactivation potential of AP-1 complexes (Yoshioka et al. 1995). Fosl1 and Fos show different yet partially overlapping expression patterns in mice. Fosl1 is expressed in extraembryonic tissues during mouse development and in brain, skin, and testes of adult mice (Schreiber et al. 1997, 2000). Fos is expressed in the placenta and in several organs of the embryo, including fetal liver, heart, brain, and developing bones. In adult mice, Fos is readily detectable in most tissues, with highest levels found in brain and bone (Smeyne et al. 1993). Gain-of-function experiments have shown that overexpression of Fra-1 leads to increased bone formation and osteosclerosis (Jochum et al. 2000), whereas overexpression of c-Fos results in osteo- and chondrosarcomas (Ruther et al. 1987; Wang et al. 1992). Targeted disruption of the Fosl1 gene leads to early embryonic lethality due to impaired vascularization of the placenta (Schreiber et al. 2000). In contrast, lack of c-Fos results in pleiotropic defects, most importantly the bone disease osteopetrosis, due to the lack of bone-resorbing osteoclasts (Johnson et al. 1992; Wang et al. 1992; Grigoriadis et al. 1994). Furthermore, c-Fos-deficient photoreceptor neurons in the retina are resistant to light-induced apoptosis (Hafezi et al. 1997; Wenzel et al. 2000). These results suggest that c-Fos and Fra-1 have distinct functions in mice. On the other hand, Fosl1 is a transcriptional target of c-Fos in osteoclast progenitors, and overexpression of Fra-1 by viral gene transfer in vitro or in transgenic mice was able to rescue c-Fos-dependent osteoclast differentiation (Matsuo et al. 2000). To test whether Fra-1 can take over c-Fos functions under physiological conditions, knock-in mice were generated in which Fos is deleted and replaced by Fosl1. Interestingly, Fosl1 can fully complement for the lack of c-Fos in bone development and substitutes for the pro-apoptotic function of c-Fos in photoreceptor neurons. Rescue of bone cell differentiation, but not photoreceptor apoptosis, is gene dosage dependent. Furthermore, Fra-1 fails to rescue the expression of c-Fos target genes in fibroblasts. These data suggest that transactivation by c-Fos might be dispensable in vivo and that stage- and cell-specific modulation of expression determines the specific functions of these genes.

For several decades, basic research on acquired and inherited retinal degeneration was substantially based on a variety of animal models, most of them originating from spontaneous mutations, others induced by damaging external agents. In the past few years, however, progress in genetic engineering has led to a rapidly growing number of transgenic animals, mostly mice, carrying constructs that lead to disruption or overexpression of candidate genes for retinal degenerations. On the one hand, these new models constitute a powerful and adaptable tool to investigate the role of specific gene mutations and the resulting cellular defects that finally lead to photoreceptor cell death. On the other hand, they extend the spectrum of animal models suitable for the newly arisen field of retinal somatic gene therapy. To assist researchers and clinicians interested in the field, this article attempts to provide a structured overview on recently developed transgenic animal models as well as on models based on spontaneous mutations and induced degenerations. In this review the authors focus on animal models for photoreceptor degeneration since the rapidly growing field of models for ganglion cell death merits its own review and would be beyond the scope of this article. Even with this restriction, the abundance of information generated especially in the past few years makes the attempt of a complete overview almost illusory. Therefore, we apologise for omissions or shortfalls extant in this review. Furthermore, we want to point out that some of the model systems described have already been used extensively. We will therefore occasionally not cite original publications but rather reviews dealing with the specific model system. Finally, we did not incorporate strategies using viral vectors and/or pharmacological substances. The specific deletion or overexpression of genes susceptible for the modulation of photoreceptor apoptosis, however, was included. The authors are aware that these …

Light-induced apoptosis of photoreceptors represents an animal model for retinal degeneration. Major human diseases that affect vision, such as age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP), may be promoted by light. The receptor mediating light damage, however, has not yet been conclusively identified; candidate molecules include prostaglandin synthase, cytochrome oxidase, rhodopsin, and opsins of the cones and the retinal pigment epithelium (PE). We exposed to bright light two groups of genetically altered mice that lack the visual pigment rhodopsin (Rpe65-/- and Rho-/-). The gene Rpe65 is specifically expressed in the PE and essential for the re-isomerization of all-trans retinol in the visual cycle and thus for the regeneration of rhodopsin after bleaching. Rho-/- mice do not express the apoprotein opsin in photoreceptors, which, consequently, do not contain rhodopsin. We show that photoreceptors lacking rhodopsin in these mice are completely protected against light-induced apoptosis. The transcription factor AP-1, a central element in the apoptotic response to light, is not activated in the absence of rhodopsin, indicating that rhodopsin is essential for the generation or transduction of the intracellular death signal induced by light.

Retinitis pigmentosa (RP) is a hereditary retinal dystrophy which leads to severe visual impairment or blindness and affects about 3.5/1000 of individuals in the industrial world. During the past decades, numerous animal models carrying mutations analogous to mutations in human RP have been studied to elucidate the molecular mechanisms leading to apoptotic photoreceptor cell death in this disease. Up to date, there is no effective treatment to influence the fatal outcome of RP. Recent progress in basic research promotes the development of new therapeutic strategies. In order to restore visual function in blind individuals, the development of electronic photoreceptor prosthesis is being investigated by several researchgroups. Other promising approaches are somatic gene therapy, the application of growth factors and/or pharmacological agents and the inhibition of photoreceptor cell death by interfering with the apoptotic pathway. However, a better understanding of the molecular events leading to cell loss due to photoreceptor apoptosis will be essential for the development of effective treatment.

The activity of transcription factors like AP-1 and Oct-1 is critical for the regulation of gene expression. Whereas Oct-1 mainly regulates the expression of housekeeping genes, AP-1 is often involved in cellular responses to external stimuli and plays an essential role in the regulation of light-induced apoptosis of mouse retinal photoreceptors. In this study, we investigated AP-1 and Oct-1 DNA binding activity and AP-1 complex composition in the mouse retina during light-induced photoreceptor apoptosis. AP-1 DNA binding activity was low in dark-adapted animals but was transiently elevated within 12 h after exposure of mice to apoptosis-inducing levels of white fluorescent light. Maximal AP-1 activity was found 6 h after light-exposure. Antibody interference analysis at 6 h after damaging light exposure and under normal light conditions revealed that the major fraction of AP-1 consists of c-Fos/JunD heterodimers in both situations. In contrast to AP-1, Oct-1 DNA binding activity was maximal in dark-adapted animals and was reduced during photoreceptor apoptosis. Transient induction of AP-1 (c-Fos/JunD) and inactivation of Oct-1 may be crucial events for light-mediated apoptosis of retinal photoreceptors.

Apoptosis is a regulated mode of single cell death that involves gene expression in many instances and occurs under physiological and pathological conditions in a large variety of systems. We briefly summarize major features of apoptosis in general and describe the occurrence of apoptosis in the retina in different situations that comprise animal models of retinitis pigmentosa, light-induced lesions, histogenesis during development, and others. Apoptosis can be separated into several phases: the induction by a multitude of stimuli, the effector phase in which the apoptotic signal is transmitted to the cellular death machinery, the excecution period when proteolytic cascades are activated, and the phagocytic removal of cellular remnants. Control mechanisms for retinal apoptosis are only beginning to be clarified. Potential apoptotic signal transducers were investigated in our laboratory, including metabolites of arachidonic acid and downstream mediators of signaling molecules such as transcription factors. Work in our laboratory revealed an essential role of the immediate-early gene product c-Fos in light-induced apoptosis. c-Fos is a member of the AP-1 family of transcription factors and, together with other members of this family, it may regulate apoptosis in the central nervous system. Expression of the c-fos gene in the retina can be evoked by light exposure and follows a diurnal rhythm. Future studies will have to clarify how light can control the expression of specific genes, and specifically, the role of c-fos and other genes of retinal apoptosis including potential target genes and signaling pathways.

Hintergrund Apoptose ist ein genregulierter Zelltod, der bei Netzhauterkrankungen wie der Retinitis pigmentosa, der Netzhautablosung sowie der proliferativen Vitreoretinopathie eine Rolle spielt. Ein besseres Verstandnis der Regulation des apoptotischen Zelltodes konnte uber eine Reduktion der Zelltodrate ein Eingreifen in den Krankheitsverlauf von Netzhauterkrankungen ermoglichen. In verschiedenen anderen Zellsystemen konnte beobachtet werden, dass oxidativer Stres uber oxidierte Arachidonsaure-Metabolite Apoptose auslosen kann. Studien in unserem Labor zeigten, dass weises Fluoreszenzlicht Apoptose in den Photorezeptoren der Rattennetzhaut in vivo auslost. Wir beobachteten zudem die Freisetzung von Arachidonsaure und die Bildung von Arachidonsaure-Metaboliten bei der Belichtung von Ratten-Netzhauten. Daher konnte die lichtinduzierte Apoptose der Photorezeptorzellen durch Arachidonsaure oder seine Metabolite vermittelt sein. Material und Methoden Isolierte Rattennetzhaute wurden mit und ohne Zusatz von 30 µmol 5S-Hydroxyperoxytetraensaure (5S-HPETE) fur 60, 120 und 180 Minuten inkubiert. Nach der Inkubation wurden die Netzhaute licht- und elektronenmikroskopisch untersucht und die Apoptoserate in den Photorezeptoren quantifiziert. Ergebnisse Der Arachidonsaure-Metabolit 5S-HPETE lost Apoptose in den Photorezeptoren der Rattennetzhaut in vitro aus. Es wurde ein signifikanter Anstieg der Apoptoserate wahrend der Inkubation mit 5S-HPETE im Vergleich zur Kontrollgruppe beobachtet. Schlusfolgerung Durch Licht freigesetzte Arachidonsaure-Metabolite stellen potentielle Botenstoffe fur die Apoptose von Photorezeptoren dar. Background Apoptosis is a gene-regulated mode of cell death which gains increasing importance in retinal pathologies such as retinitis pigmentosa, retinal detachment and pro-liverative vitreoretinopathy. A better understanding of the regulation of apoptosis could imply the means to reduce photoreceptor cell death and thereby provide therapeutic strategies to influene the time course of retinal diseases. Previous studies in our laboratory demonstrated that light induces apoptosis in the rat retina in vivo as a function of light dose. In several cell systems, oxidative stress including oxygenated metabolites of arachidonic acid (AA) was found to evoke apoptosis. We have observed a light-elicited release of AA and the subsequent formation of its metabolites in the rat retina. Therefore, AA and its metabolites appeared to be suitable candidates for the induction of apoptosis during light exposure. Materials and methods Isolated rat retinas were incubated for 60, 120 and 180 min, respectively, with and without the addition of 30 pmol 5S-hydroperoxyeicosatetraenoic acid (5-HPETE). Retinas were then processed for light- and electron microscopy and examined for the morphological signs of apoptosis. The rate of apoptosis in the outer nuclear layer was assessed quantitatively. Results 5S-HPETE induces apoptosis of photoreceptors in the rat retina in vitro. Quantitative analysis revealed a significant increase in the rate of apoptosis of 5S-HPETE-treated retinas when compared to untreated controls. Conclusion Arachidonic acid metabolites released upon light exposure may represent messenger candidates for apoptosis in the retina.

In the past few years, the interest in the research field of apoptosis in the retina has been growing rapidly. We will give a short overview of apoptosis in the context of retinal degeneration and summarize recent data obtained in our laboratory. Based on our findings, we will also discuss possible future strategies to influence apoptotic cell death in the retina and to modulate the time course of retinal dystrophies. Apoptosis is the final common pathway of photoreceptor cell death in several retinal dystrophies as well as in light-induced photoreceptor degeneration. We investigated potential signal transducers for apoptosis in our laboratory and found an essential role of the immediate-early gene product c-Fos in light-induced photoreceptor degeneration. This is of particular interest in light of the finding that c-fos is continuously upregulated concomitant with apoptotic photoreceptor death in animal models of the retinal dystrophy retinitis pigmentosa. Interference with c-fos expression or function m...

Death of retinal photoreceptors by apoptosis is observed under many physiological and pathological conditions such as histogenesis, retinal dystrophies and light-induced photoreceptor degeneration. To date, little is known about regulatory mechanisms for apoptosis in the retina. The tumor suppressor gene p53 is a regulator of apoptosis in a number of systems, however, p53-independent apoptosis has also been described. We have therefore investigated whether the lack of p53 influences the dark-adapted ERG in C57BL/6 p53−/− mice compared to p53+/+ control littermates under physiological (regular light-dark cycle) conditions. We also recorded ERGs at 12 to 14 h in darkness following diffuse bright light exposure to 8′000 or 15'000 lux for 2 h. ERG analysis over a range of 6 logarithmic units of light intensity revealed normal and virtually identical a-, b-, c-waves and oscillatory potentials in dark-adapted p53+/+ and p53−/− mice. After exposure to diffuse white fluorescent light strong decreases of all ERG components were found to be very similar in both genotypes. These data support the notion that the p53 protein is neither essential for normal retinal function nor for processes involved in light-induced depression of the ERG in mice.

Francine Behar-Cohen,1 Gilles Baillet,2 Tito de Ayguavives,2 Paula Ortega Garcia,3 Jean Krutmann,4 Pablo Peña-García,3,5 Charlotte Reme,6 James S Wolffsohn7 1French Institute of Health and Medical Research, Team 17, Centre de Recherche des Cordeliers, France; 2Research and Development Center, Essilor International, Saint Maur des Fossés, France; 3Department of Research, Fundacion Jorge Alio, Alicante, Spain; 4IUF Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany; 5Division of Ophthalmology, University Miguel Hernández, Alicante, Spain; 6Laboratory of Retinal Cell Biology, Department of Ophthalmology, University of Zurich, Switzerland; 7Life and Health Sciences, Aston University, Aston Triangle, Birmingham, UK Abstract: Ultraviolet (UV) radiation potentially damages the skin, the immune system, and structures of the eye. A useful UV sun protection for the skin has been established. Since a remarkable body of evidence shows an association between UV radiation and damage to structures of the eye, eye protection is important, but a reliable and practical tool to assess and compare the UV-protective properties of lenses has been lacking. Among the general lay public, misconceptions on eye-sun protection have been identified. For example, sun protection is mainly ascribed to sunglasses, but less so to clear lenses. Skin malignancies in the periorbital region are frequent, but usual topical skin protection does not include the lids. Recent research utilized exact dosimetry and demonstrated relevant differences in UV burden to the eye and skin at a given ambient irradiation. Chronic UV effects on the cornea and lens are cumulative, so effective UV protection of the eyes is important for all age groups and should be used systematically. Protection of children’s eyes is especially important, because UV transmittance is higher at a very young age, allowing higher levels of UV radiation to reach the crystalline lens and even the retina. Sunglasses as well as clear lenses (plano and prescription) effectively reduce transmittance of UV radiation. However, an important share of the UV burden to the eye is explained by back reflection of radiation from lenses to the eye. UV radiation incident from an angle of 135°–150° behind a lens wearer is reflected from the back side of lenses. The usual antireflective coatings considerably increase reflection of UV radiation. To provide reliable labeling of the protective potential of lenses, an eye-sun protection factor (E-SPF®) has been developed. It integrates UV transmission as well as UV reflectance of lenses. The E-SPF® compares well with established skin-sun protection factors and provides clear messages to eye health care providers and to lay consumers. Keywords: back reflection, transmission, irradiation, solar irradiance, aging, risk reduction, prevention

Apoptotic cell death in the retina was recently demonstrated in animal models of the hereditary human retinal dystrophy known as retinitis pigmentosa. Although recent evidence indicates that the proto-oncogene c-fos is a mediator of apoptosis, its precise role is unclear. In fact, under some conditions, c-fos may even protect against apoptotic cell death. In the retina, c-fos is physiologically expressed in a diurnal manner and is inducible by light. We previously observed a light-elicited, dose-dependent apoptotic response in rat photoreceptors. To determine whether c-fos is involved in the light-induced apoptotic pathway we have used control mice and mice lacking c-fos. We found that following dark adaptation and two hours of light exposure both groups of animals exhibited only a few apoptotic cells. However, at 12 and 24 additional hours after light exposure, apoptosis increased dramatically in controls but was virtually absent in those mice lacking c-fos. Therefore, c-fos is essential for light-induced apoptosis of photoreceptors. Notably, c-fos is continuously upregulated concomitant with apoptotic photoreceptor death in our system and in animal models of retinitis pigmentosa (Agarwal, N. et al., Invest. Ophthalmol. Vis.Sci. Suppl. 36, S638 and Rich, K.A. et al., Invest. Ophthalmol. Vis. Sci. Suppl. 35, 1833). Inhibition of c-fos expression might therefore represent a novel therapeutic strategy to retard the time course of retinal dystrophies and light-induced retinal degeneration.

In recent years, bright light treatment of seasonal affective disorder (SAD), recurrent depressions in fall and winter, has been discovered. Newer applications include circadian sleep phase disorder, shift work and jet lag. Apart from creating the visual signal, light can modify retinal structure and physiology. UV and visible light lead to distinct lesions of ocular tissues under certain experimental und naturalistic conditions. In light therapy, a large variety of fixtures is used but the spectral emission of lamps is mostly unknown to the user and clinician leading to the potential hazard of ocular lesions. Therefore, we have analyzed a wide selection of light sources commonly used for treatment. We measured the spectral emission and calculated irradiant doses for several light therapy regimens. Based on these measurements, potential hazards are analyzed, physiological mechanisms of light action are discussed and safety measures for bright light therapy are proposed. They include recommendations for lamps devoid of damaging spectral emissions and standardized therapy fixtures, ophthalmological monitoring of patients with eye diseases and control by optometrists for patients with healthy eyes who are likely to undergo light treatment for extended periods.

Are observations on ultraviolet (UV)- and visible light-induced ocular changes in animals relevant for human pathology? Different conclusions are drawn by different groups, depending on their perspective: while in the epidemiologist's view the evidence for those lesions is mostly limited or insufficient, laboratory scientists continually extend observations on radiation damage in animals. Consequently, there are diverging views on the necessity and specifications for eye protection. In this review, problems of epidemiological surveys and observations in humans and animal studies are discussed, and natural and artificial protection of the eye is outlined. The human and animal eye has an inherent potential for photochemical lesions due to chromophores including the visual pigments that are present at birth. Lifelong light exposure gives rise to additional absorbing molecules. With decreasing wavelengths of the electromagnetic spectrum the number of absorbing molecules rises; therefore, the likelihood of a photochemical reaction grows. As the spectral energy is augmented, more damage will occur. In our view, the knowledge gained from laboratory studies is a significant component of the total evidence from different fields —epidemiology, clinical observations, model studies and theoretical calculations — that UV radiation and short-wavelength visible light can cause acute and chronic changes in ocular structures. Such changes may comprise irreversible damage. Following recently issued recommendations of the major visual health organizations in the United States, protection against UV and blue light should be incorporated into the spectrum of safety considerations for sunglasses.

The induction of retinal degeneration by light exposure is widely used to study mechanisms of cell death. The advantage of such light-induced lesions over genetically determined degenerations is that light exposures can be manipulated according to the needs of the experimenter. Bright white light exposure can induce a synchronized burst of apoptosis in photoreceptors in a large retinal area which permits to study cellular and molecular events in a controlled fashion. Blue light of high energy induces a hot spot of high retinal irradiance within very short exposure durations (seconds to minutes) and may help to unravel the initial events after light absorption which may be similar for all damage regimens. These initial events may then induce various molecular signaling pathways and secondary effects such as lipid and protein oxidation, which may be varying in different light damage setups and different strains or species, respectively. Blue light lesions also allow to study cellular responses in a circumscribed retinal area (hot spot) in comparison with the surrounding tissue.Here we describe the methods for short-term exposures (within the hours range) to bright full-spectrum white light and for short exposures (seconds to minutes) to high-energy monochromatic blue or green light.

Light exposure not only elicits a visual response but may also alter functional and structural characteristics of the retina. Furthermore, light exposure can lead to reversible or irreversible lesions of photoreceptors and pigment epithelium. Previous studies in our laboratory have shown that light liberates arachidonic acid from retinal membrane phospholipids mainly by activating the phospholipase A2. In this study we show that light and trauma elicit the synthesis of leukotriene B4 in the isolated rat retina in vitro. Male albino rats were dark adapted for 36 h, isolated retinae were taken, incubated and exposed a) either to darkness or to 5,000 lux of cool white fluorescent light for 5, 10 or 15 min at 37 degrees C, b) either to darkness or to 5,000 lux of cool white fluorescent light for 15 min at 0 degrees C or c) either to darkness or to 5,000 lux of cool white fluorescent light for 15 min at 37 degrees C with a 5-lipoxygenase inhibitor (zileuton). Eicosanoids were extracted and leukotriene B4 levels were determined by radioimmunoassay. Removal of retinae and incubation in darkness caused a significant rise in leukotriene B4 levels with increasing incubation time. This rise was further augmented significantly after light exposure. The leukotriene B4 levels obtained when incubating the retinae either at 0 degree C or with the lipoxygenase inhibitor zileuton as well as the high specificity of the radioimmunoassay indicate that the light- and trauma-elicited synthesis of leukotriene B4 is mediated by activating the 5-lipoxygenase. Leukotriene B4 may be involved, at least in part, in the pathogenesis of retinal diseases including light damage. Curr. Eye Res. 14: 1001-1008, 1995.

The induction of retinal degeneration by light exposure is widely used to study mechanisms of cell death. The advantage of such light-induced lesions over genetically determined degenerations is that light exposures can be manipulated according to the needs of the experimenter. Bright white light exposure can induce a synchronized burst of apoptosis in photoreceptors in a large retinal area which permits to study cellular and molecular events in a controlled fashion. Blue light of high energy induces a hot spot of high retinal irradiance within very short exposure durations (seconds to minutes) and may help to unravel the initial events after light absorption which may be similar for all damage regimens. These initial events may then induce various molecular signaling pathways and secondary effects such as lipid and protein oxidation, which may be varying in different light damage setups and different strains or species, respectively. Blue light lesions also allow to study cellular responses in a circumscribed retinal area (hot spot) in comparison with the surrounding tissue.Here we describe the methods for short-term exposures (within the hours range) to bright full-spectrum white light and for short exposures (seconds to minutes) to high-energy monochromatic blue or green light.

Microglia are important components of the ocular immune system and may contribute to age-related macular degeneration. Exposure to blue light induces oxidative protein modifications similar to those observed in drusen and elicits retinal immune responses. To study the underlying cellular events, we analyzed microglial activation and monitored transcriptomic changes in blue light-induced retinal lesions. MacGreen mice with EGFP-tagged retinal microglia were exposed to blue light. At different time intervals, eyes were prepared for immunofluorescence microscopy, microarray analysis, and qRT-PCR. Retinal whole mounts and cross sections showed that EGFP-labeled microglia rapidly migrated toward the retinal lesion. Prominent transcriptomic changes occurred after 12 h, peaked at 24 h, and declined at 72 h. We identified more than 100 differentially expressed genes, including transcripts related to microglial activation, apoptosis, and regenerative signaling. A comparison of our results with published datasets from white light damage indicates overlapping but also distinct molecular mechanisms. This study extends our knowledge of transcriptomic changes in light-induced models of retinal degeneration.

Light exposure not only elicits a photic response but may also alter the metabolism and functional properties of the retina. This may be evoked by the stimulation of phospholipid derived second messenger systems. In this study, we investigated the light-evoked release of inositol 1,4,5-triphosphate in the isolated rat retina in vitro by means of high performance liquid chromatography (HPLC) detection. After prelabelling of isolated retinae with tritiated myo-inositol in darkness, they were exposed to no light or to white fluorescent light of 10,000 lux illuminance for 3,5 and 10 sec, respectively. We observed a 200% increase in the release of inositol 1,4,5-trisphosphate compared to basal values in darkness after 3 sec of light exposure with a decline after 5 sec and a return to basal values after 10 sec indicating a rapid breakdown of inositol 1,4,5-trisphosphate. Our data confirm previous studies in the amphibian retina and photoreceptors and demonstrate for the first time a light evoked inositol 1,4,5-trisphosphate release in the mammalian retina.

Using isoenzyme-specific antibodies, subtypes of protein kinase C were determined in isolated retinae of dark adapted and light-exposed rats by SDS-PAGE-Western blotting. In addition to the previously observed alpha- and beta-subspecies, the rat retina also expressed the delta-, epsilon- and zeta-isoenzymes of protein kinase C. Exposure of the animals to physiological or high levels of light does not elicit changes in the pattern or in the distribution of the different isoenzymes in the cytosolic or particulate fractions. This study demonstrates, for the first time, the presence of three calcium-independent protein kinase C isoenzymes in the rat retina.

Threads of evidence from recent experimentation in retinal morphology, neurochemistry, electrophysiology, and visual perception point toward rhythmic ocular processes that may be integral components of circadian entrainment in mammals. Components of retinal cell biology (rod outer- segment disk shedding, inner-segment degradation, melatonin and dopamine synthesis, electrophysiological responses) show self-sustaining circadian oscillations whose phase can be controlled by light-dark cycles. A complete phase response curve in visual sensitivity can be generated from light-pulse-induced phase shifting. Following lesions of the suprachiasmatic nuclei, circadian rhythms of visual detectability and rod outer-segment disk shedding persist, even though behavioral activity becomes arrhythmic. We discuss the converging evidence for an ocular circadian timing system in terms of interactions between rhythmic retinal processes and the central suprachiasmatic pacemaker, and propose that retinal phase shifts to light provide a critical input signal.

PURPOSE. In addition to photoreceptor degeneration, excessive light causes degenerative alterations in the inner retina and ganglion cell death. A disturbance in osmohomeostasis may be one causative factor for the alterations in the inner retina. Because Miiller cells mediate inner retinal osmohomeostasis (mainly through channel-mediated transport of potassium ions and water), the authors investigated whether these cells alter their properties in response to excessive blue light. METHODS. Retinas of adult rats were exposed to blue light for 30 minutes. At various time periods after treatment, retinal slices were immunostained against glial fibrillary acidic protein and potassium and water channel proteins (Kir4.1, aquaporin-1, aquaporin-4). Patch-clamp recordings of potassium currents were made in isolated Muller cells, and the swelling of Miiller cell bodies was recorded in retinal slices. RESULTS. After blue light treatment, Muller cells displayed hypertrophy and increased glial fibrillary acidic protein. The immunostaining of the glial water channel aquaporin-4 was increased in the outer retina, whereas the immunostaining of the photoreceptor water channel aquaporin-1 disappeared. Blue light treatment resulted in a decrease and a dislocation of the Kir4.1 protein in the whole retinal tissue and a decrease in the potassium conductance of Miiller cells. Hypo-osmotic stress evoked a swelling of Miiller cell bodies in light-treated retinas that was not observed in control tissues. CONCLUSIONS. The decrease in functional Kir channels may result in a disturbance of retinal potassium and water homeostasis, contributing to the degenerative alterations of the inner retina after exposure to blue light.

We have studied the effect of a dietary deprivation of n-3 fatty acids on the activity of the dopamine (DA)-de-pendent adenylate cyclase in the rat retina. Experiments were conducted in 6-month-old rats raised on semipurified diets containing either safflower oil (n-3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n-3)] in retinal phospholipids were significantly decreased in n-3 deficient rats (35–42% of control levels). This was compensated by a rise in 22:5 (n-6), the total content of poly-unsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark-adapted or light-exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light-dependent fashion. The activation was lower in rats exposed to light than in dark-adapted animals, suggesting a down-regulation of the DI DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light-exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide- and DA-dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n-3 to n-6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.

PURPOSE. Caspase-1 gene expression has been reported to be upregulated during light-induced retinal degeneration and to be reduced after neuroprotective treatments. Thus, caspase-1 may be proapoptotic in the retina. To test directly the role of caspase-1 in photoreceptor apoptosis, three mouse models were analyzed for retinal degeneration in the presence or absence of caspase-1. METHODS. Photoreceptor apoptosis was monitored in one model of induced (exposure to light) and in two models of inherited (rd1, VPP) retinal degeneration. Retinal degeneration was assessed qualitatively by light microscopy and quantitatively by the determination of free nucleosomes with ELISA or by rhodopsin measurements. Gene expression and protein levels were assessed by real-time RT-PCR and by Western blot analysis, respectively. RESULTS. Levels of caspase-1 proenzyme increased in all models of retinal degeneration concomitantly with the onset of cell death. Maturation or classic activity of caspase-1 was not detected in the retina. Ablation of caspase-1 was protective in the model of adRP (VPP mouse), but not in the two other models. Ablation of interleukin-1 receptor type 1 was without effect. Expression of monocyte chemoattractant protein (MCP)-1 increased in the model protected by caspase-1 ablation. CONCLUSIONS. Increased retinal expression of caspase-1 proenzyme may be a common marker for photoreceptor degeneration. The differential effects of caspase-1 ablation suggests a modulatory role of caspase-1 for photoreceptor apoptosis in some but not all models. Such a modulatory activity may involve a caspase-1 function different from the classic activation of interleukin-1 .( Invest Ophthalmol Vis Sci. 2006;47: 5181–5190) DOI:10.1167/iovs.06-0556

A common feature of neurodegenerative disorders is acute or progressive loss of neurons due to apoptosis. The pathological isoform of the prion protein is associated with retinal apoptosis and the cellular isoform (PrPc) has been shown to mediate protection from apoptosis in cell culture and in neonatal retinal explants. Using a model of light-induced photoreceptor apoptosis, we show in vivo that the levels of PrPc expression in the retina inversely correlate with the susceptibility of photoreceptors to light damage. Dissection of apoptotic signalling cascades suggests that PrPc acts neuroprotectively downstream of AP-1 induction. Our results reveal PrP as a neuroprotective/anti-apoptotic factor in vivo and suggest that PrPc may function as a guardian of neuronal integrity.

Human retinal dystrophies and degenerations and light-induced retinal degenerations in animal models are sharing an important feature: visual cell death by apoptosis. Studying apoptosis may thus provide an important handle to understand mechanisms of cell death and to develop potential rescue strategies for blinding retinal diseases. Apoptosis is the regulated elimination of individual cells and constitutes an almost universal principle in developmental histogenesis and organogenesis and in the maintenance of tissue homeostasis in mature organs. Here we present an overview on molecular and cellular mechanisms of apoptosis and summarize recent developments. The classical concept of apoptosis being initiated and executed by endopeptidases that cleave proteins at aspartate residues (Caspases) can no longer be held in its strict sense. There is an increasing number of caspase-independent pathways, involving apoptosis inducing factor, endonuclease G, poly-(ADP-ribose) polymerase-1, proteasomes, lysosomes and others. Similarly, a considerable number and diversity of pro-apoptotic stimuli is being explored. We focus on apoptosis pathways in our model: light-damage induced by short exposures to bright white light and highlight those essential conditions known so far in the apoptotic death cascade. In our model, the visual pigment rhodopsin is the essential mediator of the initial death signal. The rate of rhodopsin regeneration defines damage threshold in different strains of mice. This rate depends on the level of the pigment epithelial protein RPE65, which in turn depends on the amino acid (leucine or methionine) encoded at position 450. Activation of the pro-apoptotic transcription factor AP-1 constitutes an essential death signal. Inhibition of rhodopsin regeneration as well as suppression of AP-1 confers complete protection in our system. Furthermore, we describe observations in other light-damage systems as well as characteristics of animal models for RP with particular emphasis on rescue strategies. There is a vast array of different neuroprotective cytokines that are applied in light-damage and RP animal models and show diverging efficacy. Some cytokines protect against light damage as well as against RP in animal models. At present, the mechanisms of neuroprotective/anti-apoptotic action represent a "black box" which needs to be explored. Even though acute light damage and RP animal models show different characteristics in many respects, we hope to gain insights into apoptotic mechanisms for both conditions by studying light damage and comparing results with those obtained in animal models. In our view, future directions may include the investigation of different apoptotic pathways in light damage (and inherited animal models). Emphasis should also be placed on mechanisms of removal of dead cells in apoptosis, which appears to be more important than initially recognized. In this context, a stimulating concept concerns age-related macular degeneration, where an insufficiency of macrophages removing debris that results from cell death and photoreceptor turnover might be an important pathogenetic event. In acute light damage, the appearance of macrophages as well as phagocytosis by the retinal pigment epithelium are a consistent and conspicuous feature, which lends itself to the study of removal of cellular debris in apoptosis. We are aware of the many excellent reviews and the earlier work paving the way to our current knowledge and understanding of retinal degeneration, photoreceptor apoptosis and neuroprotection. However, we limited this review mainly to work published in the last 7-8 years and we apologize to all the researchers which have contributed to the field but are not cited here.