Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

hypertrophic cardiomyopathy, reflex and sensory changes, Charcot-Marie-Tooth disease, and skeletal disorders. It is hypothesized that the respiratory chain dysfunction leads to energy depletion and reactive oxygen species (ROS) accumulation which in turn produce axoplasmic stasis and swelling, thereby blocking ganglion cell function and causing loss of vision. In some patients, this loss of function is reversible in a substantial number of ganglion cells, but in others, a cell-death pathway, probably apoptotic, is activated with subsequent extensive degeneration of the retinal ganglion cell layer and optic nerve. The retinal ganglion cell degeneration and axonal loss occur predominantly in the PMB of the optic nerve.

Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress through the accumulation of ROS. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting oligodendrocytes of the optic nerves. In cell culture studies, LHON cybrid cells, constructed from the merging of heteroplasmic cytoplasm of a cell which had its nucleus removed and the cytoplasm from a second normal cell that had all its mitochondria removed, are forced by the reduced rate of glycolytic flux to utilize oxidative metabolism by a shift from glucose to galactose in the media. This causes these cells to be sensitized to a mitochondrial-mediated apoptosis. It has also been proposed that in cells carrying LHON mutations, there is a decrease in antioxidant defenses. Recent evidence shows that mitochondrial distribution reflects the different energy requirements of the unmyelinated prelaminar axons in comparison to the myelinated retrolaminar axons.

In LHON, the pathologic mutation may be either homoplasmic (involving all the mitochondria) or heteroplasmic (involving only a fraction of the mitochondria). Most heteroplasmic pedigrees have much lower penetrance but surprisingly, the disease is not milder in form.

Even with homoplasmic families, penetrance is highly variable. The rate of penetrance varies with the mutation and pedigree, although it is always greater in males. Hence, in a typical family with 11778 mtDNA, 8–10% of the women and 40–50% of the men may suffer devastating and sudden visual loss in young adulthood. This marked incomplete penetrance and gender bias imply that additional mitochondrial and/or nuclear genetic or epigenetic factors must be modulating the phenotypic expression of LHON. It is also likely that environmental factors contribute to the onset of visual failure. For example, there is increasing evidence that tobacco and alcohol consumption plays a role. There is an active search for nuclear genes that might modify the penetrance.

In LHON, fundus changes, such as microangiopathy and nerve fiber layer swelling, have been described to immediately precede or accompany the onset of visual loss (Figure 1). This process, although usually bilateral, occurs asynchronously over the course of several weeks to months and eventually evolves to severe optic atrophy and irreversible impairment of vision. The smaller caliber fibers of the PMB are selectively lost at a very early stage of the pathological process, which eventually extends to most of the rest of the nerve leading to optic atrophy.

The acute stage of LHON usually lasts a few weeks. The affected eye characteristically demonstrates an early dropout of the PMB; an edematous appearance of the rest of the nerve fiber layer, especially in the arcuate bundles; and enlarged or telangiectatic and tortuous peripapillary vessels (microangiopathy). There is absence of leakage from the optic disk or peripapillary region on fluorescein angiography These main features are seen on fundus examination, just before or subsequent to the onset of visual loss. In optical coherence tomography (OCT), LHON-affected patients showed extensive thinning of the retinal nerve fiber layer (RNFL), as would be expected in cases of optic atrophy. LHON carriers sometimes show significant RNFL thickening of the arcuate

bundles, in correlation with similar changes noted by funduscopy or the GDx nerve fiber analyzer, usually in the temporal sector, suggesting that the inferior temporal portion is affected first; this could be a subclinical sign. Several authors have described subclinical changes in the examination of asymptomatic carriers such as subtle optic disk findings, mild dyschromatopsia, OCT, and alterations in electrophysiology suggesting that LHON has some subtle chronic aspects.

In LHON-affected patients, the clinical examination reveals decreased visual acuity, dyschromatopsia, and cecocentral scotoma on visual field examination. There are a few reports of spontaneous recovery, especially with the 14484/ND6 mutation (up to 60% of cases) and in younger patients. Visual recovery may occur in one or both eyes and may happen as late as 10 years after the onset of visual loss. However, most often in LHONaffected individuals, the visual loss progresses and stabilizes within a year, with temporal optic atrophy.

There is currently no clinical evidence for an efficacious treatment to reverse vision loss in LHON. Theoretical considerations have led to the use of several agents involved with mitochondrial energy production and with anti-oxidant capabilities such as coenzyme Q10, succinate, L-carnitine, and vitamins K1, K3, C, B12, folate, and thiamine. Coenzyme Q10 is less likely to offer benefit since it is not transported to the mitochondria in sufficient concentration. A similar agent, Idebenone, has better drug delivery characteristics, and there have been a few encouraging reports and a prospective clinical trial with Idebenone in LHON. Case reports of successful recovery in LHON have to be considered in the light of spontaneous recovery. It remains unlikely that any of these agents alone or in combination will prove consistently useful in the treatment of acute visual loss in LHON or in the prophylactic therapy in asymptomatic family members at risk.

However, it is prudent to recommend the avoidance of agents that might induce oxidative stress or impair mitochondrial energy production. Most specifically, it is imperative to avoid exposure to smoke and alcohol as these environmental factors can trigger the loss of vision in susceptible individuals.

Dominant Optic Atrophy (DOA)

DOA, or Kjer’s optic neuropathy, is one of the most common forms of hereditary optic atrophies, with estimated disease prevalence in the range of 1:10 000–1:50 000. Presentation usually occurs at latency age (7–10 years old). It often presents with imperceptible onset, a slowly progressive course, and leads to mild to moderate visual impairment (20/40–20/400). Insofar as onset is insidious and progression is slow, the young patient is often unaware

of the visual loss. This usually comes to the parent’s attention after a school-based visual screening.

The inheritance of DOA is autosomal dominant. Despite the variability in expression, the penetrance is actually very high. However, the vision impairment is often very mild; hence, the apparent absence of family history may not be accurate. We recommend the direct examination of the parents. DOA presents as mild, bilateral, sometimes asymmetric loss of visual acuity. On examination, there is a central, paracentral, or cecocentral visual field deficit; temporal optic disk pallor, often with a wedge shaped area of temporal excavation (Figure 2). There is mild generalized dyschromatopsia. In general, visual prognosis is good. However, patients with DOA often re-present around the age of 35 complaining of further visual loss. In fact, this represents premature presbyopia brought about by their lifelong habit of holding reading material much closer to their faces. Therefore, Donder’s curve should not be applied to DOA patients. Instead, they should be offered plus lenses at about age 35 and graduated up to about plus 3.50 by age 50 to compensate for their closer near point.

In the year 2000 came the remarkable news that the genetic cause of dominant optic neuropathy had been identified. The gene OPA1 was, of course, nuclear and located on chromosome 3. However, the OPA1 protein encoded is imported to the mitochondria and serves structural roles in mitochondrial fission, fusion, and transport. Hence, though the genetics are somatic, the problem, like LHON, is in the mitochondria. Subsequently, other DOA genes have been found, as variations on the same theme. In addition to OPA1, there are OPA4 and other OPAs, which have been mapped to the 3q and 18q regions, respectively. All these genes are responsible for mitochondrial structural proteins.

Histological examination exhibited diffuse atrophy of the retinal ganglion cell layer, which is associated with atrophy and loss of myelin within the optic nerves. As in LHON, the retinal ganglion cells and axons lost are predominantly those of the PMB. However, the extent of axonal loss is significantly less in DOA.

In DOA, there is also an increased occurrence of associated sensorineural hearing loss so these patients should be advised to undergo audiology investigation. As in LHON, many agents have been tried as possible treatment options, and none were found to be effective. It remains unclear as to the role of environmental factors. These patients should be offered genetic counseling.

Recessive Optic Atrophy

This autosomal recessive somatic condition is the most uncommon form of inherited optic nerve disease.

Unlike LHON and DOA, ROA is usually discovered in the first 3–4 years of life. It often presents as severe visual impairment, frequently associated with searching nystagmus. Visual acuity ranges between no light perception (NLP) to 20/400. There is diffuse optic disk atrophy, sometimes with attenuation of the retinal arterioles, similar to that seen in tapetoretinal degenerations. Hence, the differential diagnosis of ROA is not so much a comparison to the other hereditary optic neuropathies as it is a comparison to retinal dystrophies. Electrophysiology using eletroretinography plays an important role in differentiating ROA from tapetoretinal degeneration, retinitis pigmentosa, or Leber’s congenital amaurosis, this is normal in ROA and severely impaired in the retinal degenerations.

The gene/chromosome for ROA remains to be identified. Not surprising for an autosomal recessive condition, there are several reports of consanguinity related to ROA.

Other Inherited Conditions with Optic Atrophy

There are several well-characterized syndromes that also include optic atrophy. Wolfram’s syndrome which is characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness is linked to the WFS1 gene located on chromosome 4p. Patients with Behr’s syndrome, which consists of progressive encephalopathy, mental retardation, ataxia, nystagmus, and pes cavus, may also have optic atrophy. It is linked to the OPA3 gene located on chromosome 19q. Friederich’s ataxia often includes vision loss and optic atrophy.

Conclusion

All of the inherited optic neuropathies serve to remind us that the optic nerve is highly dependent on mitochondrial

function. Indeed, acquired optic neuropathies that affect mitochondrial metabolism are often in the differential diagnosis of genetic optic neuropathies. The genetics of LHON involve a mutation of mitochondrial DNA resulting in impairments of complex I, which in turn leads to decreased ATP production and increased ROS. This is in contradistinction to DOA in which the genetics involve nuclear DNA. However, despite the fact that these mutations are somatic, the OPA genes control structural mitochondrial proteins that also limit mitochondrial functions such that the final consequences are similar: decreased ATP production and increased ROS. Hence, inherited optic neuropathies and metabolic optic neuropathies, whether toxic or nutritional, all share the common pathophysiology of producing mitochondrial dysfunction and subsequent retinal ganglion cell loss. Not surprisingly, all mitochondrial optic neuropathies share a similar clinical presentation. If a treatment proves to be effective in one, it is likely to be of benefit in the other mitochondrial optic neuropathies.

Exciting new treatment options with agents that modulate mitochondrial function that have anti-apoptotic effects or are helpful for neuronal survival, are currently being studied. Inherited optic neuropathies may serve as the ideal model with which to test these purportive neuroprotective agents.

Further Reading

Barboni, P., Savini, G., Valentino, M. L., et al. (2005). Retinal nerve fiber layer evaluation by optical coherence tomography

in Leber’s hereditary optic neuropathy. Ophthalmology 112(1): 120–126.

Carelli, V., Ross-Cisneros, F. N., and Sadun, A. A. (2002). Optic nerve degeneration and mitochondrial dysfunction: Genetic and acquired optic neuropathies. Neurochemistry International 40(6): 573–584.

Carelli, V., Ross-Cisneros, F., and Sadun, A. (2004). Mitochondrial dysfunction as a cause of optic neuropathies. Progress in Retinal and Eye Research 23: 53–89.

Chalmers, R. M. and Schapira, A. H. V. (1999). Clinical, biochemical and molecular genetic features of Leber’s hereditary optic neuropathy.

Biochimica et Biophysica Acta 1410: 147–158.

Newman, N. J. (1998). Hereditary optic neuropathies. In Miller, N. R. and Newman, N. J. (eds.) Walsh and Hoyt’s Clinical NeuroOphthalmology, pp 742–756. Baltimore: Williams and Wilkins.

Riordan-Eva, P., Sanders, M. D., Govan, G. G., et al. (1995). The clinical features of Leber’s hereditary optic neuropathy defined by the presence of a pathogenic mitochondrial DNA mutation. Brain 118(2): 319–337.

Sadun, A. A. (2002). Metabolic optic neuropathies. Seminars in Ophthalmology 17(1): 29–32.

Sadun, A. A., Carelli, V., Salomao, S. R., et al. (2003). Extensive investigation of large Brazilian pedigree of Italian ancestry (SOA-BR) with 117788/Haplogroup J Leber’s hereditary

optic neuropathy (LHON). American Journal of Ophthalmology

136: 231–238.

Sadun, A., Salomao, S. R., Berezovsky, A., et al. (2006). Subclinical carriers and conversions in Leber’s hereditary optic neuropathy:

A prospective psychophysical study. Transactions of the American Ophthalmological Society 104: 51–61.

Sanchez, R. N., Smith, A. J., Carelli, V., et al. (2006). Leber’s hereditary optic neuropathy possibly triggered by exposure to tire fire. Journal of Neuroophthalmology 26(4): 268–272.

Smith, J. L., Hoyt, W. F., and Susac, J. O. (1973). Ocular fundus in acute Leber optic neuropathy. Archives of Ophthalmology 90(5): 349–354.

Nikoskelainen, E. K. (1994). Clinical picture of LHON. Clinical Neuroscience 2: 115–120.

Wallace, D. C., Singh, G., Lott, M. T., et al. (1988). Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242: 1427–1430.

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Glossary

Acute light damage – Single exposure of intense light (1000–13 000 lux) for a brief period (20 min–24 h), which causes photoreceptor cell death, mainly in the central retina.

Apoptosis – A form of programmed cell death in multicellular organisms, which involves a series of biochemical events leading to a characteristic cell morphology and death. Death and the disposal of cellular debris do not damage the neighboring cells. Light-induced photoreceptor cell death occurs by apoptosis.

Chronic light damage – Exposure to sublethal doses of light (200–800 lux) in a cyclic manner (day–night) for a longer period of time (7–12 weeks). This causes overall thinning of the photoreceptor layer in the entire retina.

Inflammation – The complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, or irritants. It is a protective response by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue.

Photoisomerization – The event in which a lightsensitive molecule absorbs a photon of light energy and undergoes a chemical change. In the current context, the 11-cis-retinal chromophore of rhodopsin is isomerized to all-trans-retinal, leading to activation of rhodopsin (R*) and initiation of the visual process. Photostasis – Retinal adaptive response to different levels of cyclic illuminances. In bright cyclic light, rod outer segment (ROS) length is shortened, concentration of rhodopsin per unit of ROS is decreased, and ROS membranes become disorganized, compared to animals raised in dim cyclic light. This remarkable plasticity allows the retina of animals raised in dim and bright cyclic light to catch an equivalent number of photons each day.

Introduction

In 1966, Werner Noell discovered that the exposure of albino rats to bright visible light led to a rapid, specific, and irreversible loss of rod photoreceptor cells. Over the

next four decades, this light-damage model has been used extensively to study mechanisms of retinal cell death and to test the efficacy of a variety of putative neuroprotective compounds. In his pioneering work, Noell discovered that photo-bleaching of rhodopsin was absolutely essential for light damage; rats raised on vitamin-A-deficient diets were not sensitive to light damage and the susceptibility to light damage followed the absorption spectrum of the visual pigment rhodopsin. Although the mechanism for initiation of the cell death signal in light damage is not known, recent studies have shown that while activation of rhodopsin is required, the visual transduction pathway downstream is not. Several outstanding reviews have been written on various aspects of light damage and therapeutic interventions. This article focuses on recent advances in identifying potential molecular mechanisms of cell injury and death, as well as the discovery of two independent mechanisms of endogenous self protection which are activated by light and stress in retinal photoreceptors.

There are several major advantages in using light damage to study retinal degeneration. Most inherited retinal degenerations occur over a long period of time with only a few photoreceptors dying each day. This makes it extremely difficult to biochemically study mechanisms of cell death or to study the effectiveness of therapeutic interventions. Light damage, on the other hand, has the advantage to more or less induce cell death in whole populations of photoreceptors at once. In addition, light damage can be tuned in terms of severity by varying the intensity of the light and duration of exposure. Hence, the intensity and duration required to cause a certain level of damage are correlated and a longer exposure can substitute for a higher intensity.

Over the years, two models of light damage have emerged: acute (short bright exposure) and chronic (less bright cyclic exposure). Both lead to retinal degeneration, but have quite different phenotypes. In the acute model, animals placed in light intensities ranging from 1000 to 13 000 lux in a time frame of 20 min to 24 h undergo a rapid and specific loss of rod photoreceptors. An example of acute light damage in a Sprague Dawley (SD) rat is shown in Figure 1. There is a clear demarcation of photoreceptor cell death, with normal-appearing retina (yellow arrow) adjacent to an area of moderate-to-massive cell death (white arrows). It is interesting that the subretinal space underlying the normal retina (yellow arrow) has been infiltrated with immune cells, suggesting that an inflammatory process is occurring prior to photoreceptor cell death. The role of inflammation in light damage is

discussed subsequently in this article. A so-called spider graph of the thickness of the outer nuclear layer (ONL, rod nuclei) measured at defined distances from the optic nerve head to the inferior and superior ora serrata along the vertical meridian clearly demonstrates the specific loss of photoreceptor cells in the central superior region of the retina (Figure 2). The chronic light damage model involves raising albino rodents in sublethal levels of cyclic light (200–800 lux, depending upon the strain). Retinas of SD rats raised from birth to 7 weeks of age in 700-lux cyclic light show remarkably normal retinal morphology except for a thinning of the ONL (Figure 3). The loss of

photoreceptor cells is quite different from that which occurs in acute light damage, showing no regional differences (Figure 4). An interesting feature of the chronic light damage model is that, although the retina slowly loses photoreceptors, it is protected against the massive loss that occurs when these animals are placed in acute bright light. This point is discussed in detail later on in this article. These two models thus provide a means of testing putative neuroprotective compounds in two experimental paradigms. They are also useful to identify the molecular mechanisms that are responsible for lightinduced damage, although the initial sequence of events leading to apoptosis may differ significantly from one protocol to another.

Acute light damage

Figure 1 Sprague-Dawley rats were born and raised in 5 lux cyclic light. At 6–7 weeks of age, they were exposed to 2700-lux light for 6 h, after which they were returned to their cyclic light environment for 7 days. The white arrows point to areas of moderate to severe degeneration adjacent to an area of normal retina (yellow arrow).

Role of Rhodopsin Activation in Light

Damage

While the precise mechanisms for cell damage are not fully understood, several genes have been identified that regulate photoreceptor sensitivity to light damage. When first identified, the action spectrum of light damage overlapped with the absorption spectrum of rhodopsin, suggesting a role for photoisomerization and phototransduction in cell injury. Indeed, a single, strong rhodopsin bleaching event is sufficient to induce light damage. The role of photoisomerization of 11-cis-retinal and rhodopsin activation as a requirement for light damage has been demonstrated pharmacologically and genetically. Pharmacological inhibition of rhodopsin regeneration with either the anesthetic halothane or by retinoic acid analogs, isotretinoin or 13-cis- retinoic acid, renders retinal photoreceptors less susceptible to light damage. Therefore, the severity of damage depends on the regeneration of rhodopsin during light exposure.

This sensitivity may be affected by polymorphisms or disease-causing mutations in opsin. The P23H opsin mutation is known to cause retinitis pigmentosa (RP) in humans. Work by Muna Naash and Daniel Organisciak, using transgenic mice or rats expressing human rod opsin with the P23H mutation, has shown that photoreceptors from these animals degenerate faster in bright cyclic light. Several recent studies have shown that mice with mutations in opsin, and phototransduction termination proteins arrestin and rhodopsin kinase are more sensitive to light damage. These results suggest that a prolonged R* or activated rhodopsin state is responsible for sensitivity to light damage. In more recent years, the Zurich group of Charlotte Reme, Andreas Wenzel, and Christian Grimm confirmed the

Chronic light damage

Figure 3 Sprague-Dawley rats were born and raised under 700-lux cyclic light until they were 7 weeks old. The retinas appear normal except for the thinning of the outer nuclear layer (ONL; yellow arrow). ONH, optic nerve head.

necessity for rhodopsin photobleaching by demonstrating that mice lacking the RPE65 gene, which encodes a protein necessary for photoisomerization of all-trans-retinyl esters, were protected from light-induced cell death. The same group has also shown that a slow rate of rhodopsin regeneration due to an L450M variation in the RPE65 protein also renders mice less susceptible to light damage. However, while activation of rhodopsin is required for acute light damage, activation of phototransduction through guanine nucleotide-binding protein at (Gat or transducin) is not. This was demonstrated in an elegant study by Wenshan Hao using combinations of rhodopsin kinase, arrestin, and transducin knockout mice. The study clearly suggests that a transducin-independent but, rhodopsin-dependent, signaling is required for acute light damage.

Mechanisms of Photic Injury: From Gene Expression to the Molecular Pathway

Gene expression analyses by DNA microarray studies have uncovered several possible molecular mechanisms of cell death in light damage. Our primary focus has been acute light damage (2700–3000 lux for 6 h) in albino mice or rats. This exposure paradigm induces oxidative stress that is sufficient to cause loss (>80%) of the photoreceptor cells in the central retina with no recoverable electroretinogram (ERG). However, cell death does not occur immediately following light exposure. It should be noted that by isolating RNA immediately after light exposure one can assess the stress response of photoreceptors before the onset of cell loss and detectable apoptotic cells in the retina. Therefore, by determining this early response one can decipher the molecular pathways that might regulate light-induced cell death. Retinas harvested

immediately after the light exposure were used to generate complementary DNA (cDNA) to expressed messenger RNA (mRNA). The expression of the genes is studied by high-throughput DNA microarray analysis followed by quantitative (real-time) RT-PCR methods (RT-PCR, reverse transcriptase-polymerase chain reaction).

Apoptosis Genes

It has been well documented that apoptosis is the mechanism of photoreceptor cell death in light damage. Light damage induces expression of members of the activator protein 1 (AP-1) transcription factor family: c-fos, FosL1 (fra-1), junB, and c-Jun. The dimeric AP-1 transcription factor is formed by either c-fos or fra-1 binding to Jun proteins. The bright-light-induced apoptosis in retina is dependent upon activation of c-fos, and this c-fos upregulation plays a critical proapoptotic role, as evidenced from c-fos knockout mice, which showed marked resistance to light damage. Further, activation of the glucocorticoid receptor, which inhibits AP-1, also protects against light damage.

There are indications of involvement of other apoptosis genes in light damage. We have observed marked upregulation in the expression of tumor necrosis factor (TNF) receptor superfamily, member 1A (Tnfrsf1a), growth arrest and DNA-damage-inducible, beta (Gadd45b), and pleckstrin homology-like domain, family A, member 1 (Phlda1) genes. The protein encoded by Tnfrsf1a is one of the major receptors for the TNF-alpha (TNFa). This receptor can activate nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), mediate apoptosis, and function as a regulator of inflammation. In addition, the anti-apoptotic protein BCL2-associated athanogene 4 (BAG4/SODD) and adaptor proteins TRADD and TRAF2 have been shown to interact with this receptor. GADD45b can respond to environmental stresses by mediating activation of the p38/JNK pathway, which is involved in the regulation of growth and apoptosis. Phlda1 encodes an evolutionarily conserved proline–histidine-rich nuclear protein, which may play an important role in the antiapoptotic effects of insulin-like growth factor-1. In summary, we observed a significant upregulation of many proapoptotic genes, which are mainly involved in the AP-1-mediated apoptotic pathway and did not detect any changes in the caspase-related genes in the initial phase of degeneration.

Role of Oxidant Stress in Light Damage

Oxidant stress has long been implicated in the pathogenesis of light damage. Intense light involves the generation of oxidants in the photoreceptors and the accumulation of oxidatively modified lipids, nucleic acids, and proteins. Oxidant stress is further supported by several reports describing protection from light damage induced by a

variety of antioxidants, including ascorbate, dimethylthiourea, thioredoxin, and NG-nitro-L-arginine-methyl ester (L-NAME). We observed that pretreatment with the free radical trap phenyl-N-tert-butylnitrone (PBN) completely abrogates light-induced degeneration of the retina. The PBN protection not only involves the stabilization of the free radicals, but also suppresses the expression of many proinflammatory and apoptotic genes. Gene expression analysis has shown that light damage upregulates the expression of antioxidant genes heme oxygenase 1 (Ho-1), superoxide dismutase (Sod), thioredoxin, glutathione peroxidase, ceruloplasmin (Cp), and metallothioneins (Mt)-1 and-2. Mice expressing mutant SOD1 are highly susceptible to light damage and have accelerated age-dependent degeneration of the retina. CP is a ferroxidase that functions as an antioxidant by oxidizing iron from its ferrous to ferric form. Ironderived hydroxyl radicals produced by the Fenton reaction may be important mediators of retinal photic injury as systemic administration of the iron chelating reagent desferrioxamine attenuates light-induced damage in rat retinas. Metallothioneins (MTs), another group of antioxidants, are copperand zinc-binding proteins which can quench superoxide and hydroxyl radicals. Expression of oxidant defense genes further supports the hypothesis that light-induced retinal degeneration involves oxidative stress.

Role of Inflammation in Light Damage

We have noticed recurrent appearance of a host of inflammatory genes in the group of upregulated genes in many light-damage studies. In fact, invading immune cells are often observed in the retina near dying photoreceptors. These can be seen under the relatively normal photoreceptors (yellow arrow, Figure 1) adjacent to the injured photoreceptors (white arrows, Figure 1). While the connection between inflammation and light damage is not well known, two recent studies have suggested there is a strong link. Work from Barbel Rohrer has shown that genes related to complement activation are upregulated in response to light stress and that elimination of complement factor D reduces susceptibility to light damage. Work from Ying-qin Ni has shown that light stress leads to the upregulation of interlukin 1b (IL-b) and activation of resident microglia. Significantly, the study found that inhibiting microglia activation with naloxone protected photoreceptors from subsequent light damage. These studies suggest that an inflammatory response plays an active role in promoting photoreceptor death in light damage. Inflammation is also becoming recognized as a key factor in many retinopathies, including the epidemic forms of diabetic retinopathies and age-related macular degeneration (AMD). Therefore, a thorough investigation into the inflammatory process in the retina is essential for an understanding of the mechanism of light damage as well as other forms of retinal degeneration.

Light damage rodent models are slowly being recognized as models for retinal inflammation and are suitable models for human AMD. Here we summarize our observation of inflammatory gene expression changes. Photic injury upregulates the expression of many chemokine genes, Ccls (Ccl2, Ccl3, Ccl4, and Ccl7) and Cxcls (Cxcl1, Cxcl11, Cxcl10, and Cxcl9). Chemokines are a group of small (8–14 kDa), mostly basic, structurally related molecules that regulate cell trafficking of various types of leukocytes through interactions with a subset of guanine nucleotide-binding protein (G-protein-coupled) receptors. These molecules are divided into two major subfamilies, CXC and CC, based on the arrangement of the first two of four conserved cysteine residues; the two cysteines are separated by a single amino acid in CXC chemokines and are adjacent in CC chemokines. Chemokines also play fundamental roles in the development, homeostasis, and function of the immune system, and they have effects on cells of the central nervous system as well as on endothelial cells involved in angiogenesis or angiostasis. Ccl2 or monocyte chemoattractant protein-1 (Mcp-1) upregulation is very robust and very early in the process of acute light damage. They may signal injury and recruit choroidal macrophages to scavenge retinal debris. Homozygous deletion of Ccl2 resulted in a mouse phenotype reported to be similar to human AMD. CCL3, also known as macrophage inflammatory protein-1, or monokine, is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes. CCL4, a cytokine that is upregulated during the inflammatory response, is involved in the recruitment of neutrophils. CCL7 or monocyte chemotactic protein 3, a secreted chemokine, attracts macrophages during inflammation and metastasis. In addition, CXCL10 binds to its receptor CXCR3 and results in pleiotropic effects, including stimulation of monocytes, migration of natural killer and T-cells, and modulation of adhesion molecule expression. Upregulation of CXCL11 indicates probable activation of interferon gamma, which is a potent inducer of CXCL11 transcription.

Besides chemokines, we found significant upregulation of genes, which are either members of classic inflammatory proteins or involved in regulation of cellular inflammation. Expression of intercellular adhesion molecule 1 (Icam1), CCAAT/enhancer-binding protein (Cebpb/C/ EBP, beta), cytokine-cardiotrophin-like cytokine factor 1 (Clcf1), lipopolysaccharide-induced TNF factor (Litaf ), cyclooxygenase 2 (Cox2), ring finger protein 125 (Rnf 125), and Cd44 genes were significantly upregulated in acute light stress. The protein encoded by the Cebpb gene is a basic-leucine zipper (bZIP) transcription factor, which can bind as a homodimer to certain DNA regulatory regions of genes involved in immune and inflammatory responses and has been shown to bind to the IL-1 response element in the IL-6 gene, as well as to regulatory

regions of several acute-phase and cytokine genes. The CLCF1 protein belongs to the IL-6 family of cytokines, which are involved in cell signaling through phosphorylation of gp130. Lipopolysaccharide is a potent stimulator of monocytes and macrophages, causing secretion of TNFa and other inflammatory mediators. COX2 is the key enzyme in prostaglandin biosynthesis and acts both as a dioxygenase and as a peroxidase and is involved in inflammation and mitogenesis. The Rfn125 gene encodes a novel E3 ubiquitin ligase that contains an N-terminal RING finger domain, which may function as a positive regulator in the T-cell receptor signaling pathway. This is a small list of the inflammatory genes ( 30% of all the upregulated genes) that are induced in acute light damage and clearly indicates a role in the pathogenesis of light damage.

Tissue Remodeling

Inflammatory signaling events are usually followed by tissue remodeling for the invasion of the macrophages and other cells, which then leads to the process of advanced cell death and removal of debris. We observed significant upregulation of matrix metallopeptidase 3 (Mmp3), tissue inhibitor of metallopeptidase 1 (Timp1), growth differentiation factor 15 (Gdf15), and plasminogen activator, tissue (Plat) genes. MMP3 is an endopeptidase that degrades extracellular matrix proteins and TIMP1 acts as an inhibitor of metalloprotease activity; PLAT is an enzyme that also plays a role in cell migration and tissue remodeling.

Transcription Factors

Many of the genes described under apoptosis, oxidative stress, and inflammation are transcription factors. However, there are other transcription factors upregulated in retinal light damage, which are involved in neuronal injury and various processes in cell death and survival. These include early growth response 1 (Egr1), zinc finger protein 36 (Zif36), activating transcription factor 3 (Atf3), and stress-signal transducer and activator of transcription 3 (Stat3).

Identification of Two Nonredundant

Mechanisms of Endogenous Protection of

Photoreceptors

Several recent studies have been designed to reveal pathways that protect photoreceptors from light damage. These studies have identified two independent protective pathways. One pathway is rapidly activated by phosphorylation

following light stimulation, while the second pathway requires induction of new gene expression in response to chronic light stress.

Rhodopsin-Activated Endogenous Protection

Work from Raju Rajala has demonstrated that rhodopsin activation by light results in ligand-independent activation of the insulin receptor, which leads to activation of downstream signaling including increased PI3K and Akt kinase activity in photoreceptors. This activation was subsequently shown to be transducin independent. Genetic inactivation of the insulin receptor, insulin receptor substrate 2 (IRS2), AKT2, or BCL-XL have all led to increased photoreceptor susceptibility to acute light damage. These studies suggest that rhodopsin signaling, independent of transducin, is responsible for the activation of a defense mechanism through the insulin receptor and AKT2. Insulin receptor regulation of AKT2 is an ideal protective pathway for acute changes in light intensity since the entire pathway can be activated quickly through a series of phosphorylation events and does not require new gene expression. However, the AKT2 defense mechanism is overwhelmed as the duration of light exposure or intensity of light begins to induce cell death. It seems that the retina requires a secondary system of protection from chronic stresses such as prolonged bright light exposure and inherited genetic mutations.

Identification of Mechanisms for Chronic Light Stress-Induced Endogenous Protection

Noell’s observation that animals raised in bright cyclic light were less susceptible to a subsequent light challenge than those raised in dim cyclic light or darkness inspired the discovery by John Penn and Ted Williams in the 1980s that albino rats born and raised in bright (but sublethal) cyclic light were protected from acute light damage. They discovered an enormous plasticity of the retina. Animals raised in dim cyclic light enhanced their chances of photon capture by lengthening their outer segments and increasing the packing density of rhodopsin in rod outer segment disk membranes. On the other hand, animals raised in relatively bright cyclic light reduced the length of outer segments, which also became somewhat disorganized, and decreased the packing density of rhodopsin in the disk membrane. The net result was to reduce the efficiency of photon capture by rhodopsin. Penn and Williams coined the term photostasis to describe the phenomenon of biochemical and morphological adaptation of the retina to modify efficiency of photon capture in animals exposed to different levels of cyclic light. It was suggested that these changes allowed photoreceptors to capture an equivalent number of photons each day regardless of their light environment. The morphological

changes associated with bright cyclic rearing were also accompanied by biochemical changes described by Penn and Anderson that included increased activity of glutathione enzymes (peroxidase, S-transferase, and reductase), elevation of retinal vitamins E and C, and decreased levels of polyunsaturated fatty acids (substrates for lipid peroxidation). These studies suggest that in response to chronic light stress, photoreceptors undergo photostasis to reduce activation of rhodopsin and induce an antioxidant defense. Similar findings have been shown in both mice and rats. Importantly, these stress-induced molecular and morphological adaptations were shown to protect photoreceptors almost completely from acute light damage.

The mechanism by which retinas of rodents raised in bright cyclic light are protected from acute light damage is an important area of research and significant progress has been made toward identifying the factors and receptors that are required, as well as identifying their relevant signal transduction pathways. Since the insulin receptor/ PI3K/Akt2 pathway is involved in providing protection from acute light damage, this pathway was considered a likely candidate for chronic light stress-induced protection. However, recent findings suggest that chronic light stress-induced protection is independent of this pathway. Unlike the insulin receptor and AKT2-dependent protection, induced protection requires several days of preconditioning, suggesting that new gene expression is required. This indirectly suggests a different mechanism. In support of this, we have shown that induced protection is independent of AKT phosphorylation, and we have shown that Akt2 knock-out mice have induced protection that is identical to wild-type mice. The insulin receptor and AKT2 appear to function as a rapid response to an acute injury, but is not necessary or perhaps is unable to protect from chronic injury. These studies demonstrate that there are two mechanisms of endogenous protection, one for acute injury that utilizes the insulin receptor and AKT2, and one for induced protection from chronic injury.

Role of Leukemia Inhibitory Factor

Several groups, including Steinberg, LaVail, Wen, and Stone, have shown that light preconditioning in rats induces the expression of several factors including basic fibroblast growth factor (FGF) (FGF2) and ciliary neurotrophic factor (CNTF). More recently in mice, the Grimm and Ash laboratories have shown that preconditioning induces the expression of leukemia inhibitory factor (LIF), cardiotrophin-like ligand (CLC), onchostatin M (OSM), FGF2, endothelin 2 (End2), and brainderived neurotrophic factor (BDNF). While many factors are upregulated, it was not known which were responsible for induced protection. Early work from Steinberg and LaVail demonstrated that the intravitreal injection of FGF2, CNTF, LIF, or BDNF resulted in substantial