Light Damage Models of Retinal Degeneration.

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 damage as a model of retinal degeneration.

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.

Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.

BACKGROUND: Optical 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 FINDINGS: We 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/SIGNIFICANCE: We 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.

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

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.

Müller cell response to blue light injury of the rat retina.

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 Müller 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 Müller cells, and the swelling of Müller cell bodies was recorded in retinal slices. RESULTS: After blue light treatment, Müller 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 Müller cells. Hypo-osmotic stress evoked a swelling of Müller 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.

Localization of glial aquaporin-4 and Kir4.1 in the light-injured murine retina.

Excessive light causes damage to photoreceptor and pigment epithelial cells, and a local edema in the outer retina. Since Müller 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 Müller cells. Mice were treated with bright white light (intensity, 15,000lx) for 2h. Light treatment results in Müller 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 Müller 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 Müller 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.

R91W mutation in Rpe65 leads to milder early-onset retinal dystrophy due to the generation of low levels of 11-cis-retinal.

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.

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light.

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.

Molecular mechanisms of light-induced photoreceptor apoptosis and neuroprotection for retinal degeneration.

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.

New retinal light damage QTL in mice with the light-sensitive RPE65 LEU variant.

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.

The genetic modifier Rpe65Leu(450): effect on light damage susceptibility in c-Fos-deficient mice.

PURPOSE: To test whether introduction of the Rpe65Leu(450) variant can overcome protection against light-induced photoreceptor apoptosis in mice without the activator protein (AP)-1 constituent c-Fos. METHODS: c-Fos-deficient mice (c-fos(-/-)) carrying the Leu(450) variant of RPE65 were compared with c-fos(-/-) mice with Rpe65Met(450). Expression of RPE65 was analyzed by Western blot analysis. Rhodopsin regeneration was determined by measuring rhodopsin after different times in darkness after bleaching. Susceptibility to light-induced damage was tested by exposure to white light and subsequent morphologic analysis. Activation of AP-1 and its complex composition was analyzed by electromobility shift assay (EMSA) and antibody interference. The contribution of AP-1 to apoptosis was tested by pharmacological inhibition of AP-1, using dexamethasone. RESULTS: Compared with RPE65Met(450), introduction of the RPE65Leu(450) variant led to increased levels of RPE65 protein, accelerated rhodopsin regeneration, loss of protection against light-induced damage, and AP-1 responsiveness to toxic light doses, despite the absence of c-Fos. c-Fos was mainly replaced by Fra-2. Application of dexamethasone restored resistance to light-induced damage. CONCLUSIONS: Increasing retinal photon catch capacity by introducing the Rpe65Leu(450) variant overcomes light damage resistance provided by c-fos deficiency. Thus, a variation of RPE65 at position 450 is a strong genetic modifier of susceptibility to light-induced damage in mice. Under conditions of high rhodopsin availability during exposure to light, Fra-2 and, to a minor degree, FosB substitute for c-Fos and enable light-induced AP-1 activity and thus photoreceptor apoptosis. Regardless of the AP-1 complex's composition, glucocorticoid receptor activation inhibits AP-1 and prevents apoptosis. Thus, not the absence of c-Fos per se, but rather impairment of AP-1 DNA binding is protective against light-induced damage. This impairment may result from the absence of c-Fos or glucocorticoid receptor-mediated transrepression.

Evidence for two apoptotic pathways in light-induced retinal degeneration.

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.

HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced 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.

Fra-1 substitutes for c-Fos in AP-1-mediated signal transduction in retinal apoptosis.

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.

Localization of organic anion transport protein 2 in the apical region of rat retinal pigment epithelium.

PURPOSE: The organic anion transporting protein (Oatp)-2 has been cloned from brain and retina. It mediates transport of many endogenous and exogenous amphiphilic compounds across the plasma membrane in a sodium-independent manner. In the brain it resides at the luminal and abluminal membrane of the capillary endothelium and at the basolateral membrane of the choroid plexus epithelium. In the liver, it is expressed at the basolateral membrane of hepatocytes. Its exact localization and function in the retina are unknown. Therefore, the purposes of the present study were to determine the cellular and subcellular localization and the potential functional aspects of Oatp2 in the retina. METHODS: Oatp2 was detected in rat retinal tissue by immunofluorescence confocal microscopy and by Western blot analysis, with a specific antibody. A Xenopus laevis oocyte expression system was used for functional transport studies. RESULTS: Oatp2 immunoreactivity was abundantly present at the apical microvilli of the rat retinal pigment epithelium and to a lesser degree in small retinal vessels. In the oocyte expression system, N-retinyl-N-retinylidene ethanolamine (A2E), an unusual cationic, amphiphilic retinoid, exhibited competitive Cis inhibition of Oatp2-mediated digoxin transport with an estimated K(i) of approximately 37 microM. CONCLUSIONS: In rat retina, Oatp2 is localized at the interface between the pigment epithelium and the photoreceptor outer segments. A2E is a competitive inhibitor of Oatp2-mediated substrate transport, suggesting that A2E or A2E-like compounds and some retinoids may be substrates for Oatp2 transport.

Apoptosis in the Retina: The Silent Death of Vision.

Pathogenetic mechanisms of retinal degeneration include cell loss by apoptosis. This gene-regulated mode of single-cell death occurs in a number of widespread human diseases such as neurodegeneration. The knowledge of genes and signaling in retinal apoptosis is expanding and opens up therapeutic strategies to ameliorate blinding retinal diseases.