Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

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Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription–PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.

Natural Compounds from Saffron and Bear Bile Prevent Vision Loss and Retinal Degeneration

All retinal disorders, regardless of their aetiology, involve the activation of oxidative stress and apoptosis pathways. The administration of neuroprotective factors is crucial in all phases of the pathology, even when vision has been completely lost. The retina is one of the most susceptible tissues to reactive oxygen species damage. On the other hand, proper development and functioning of the retina requires a precise balance between the processes of proliferation, differentiation and programmed cell death. The life-or-death decision seems to be the result of a complex balance between pro- and anti-apoptotic signals. It has been recently shown the efficacy of natural products to slow retinal degenerative process through different pathways. In this review, we assess the neuroprotective effect of two compounds used in the ancient pharmacopoeia. On one hand, it has been demonstrated that administration of the saffron constituent safranal to P23H rats, an animal model of retinitis pigmentosa, preserves photoreceptor morphology and number, the capillary network and the visual response. On the other hand, it has been shown that systemic administration of tauroursodeoxycholic acid (TUDCA), the major component of bear bile, to P23H rats preserves cone and rod structure and function, together with their contact with postsynaptic neurons. The neuroprotective effects of safranal and TUDCA make these compounds potentially useful for therapeutic applications in retinal degenerative diseases.

Mild systemic hypoxia and photopic visual field sensitivity

Purpose: Flickering stimuli increase the metabolic demand of the retina,making it a sensitive perimetric stimulus to the early onset of retinal disease. We determine whether flickering stimuli are a sensitive indicator of vision deficits resulting from to acute, mild systemic hypoxia when compared to standard static perimetry. Methods: Static and flicker visual perimetry were performed in 14 healthy young participants while breathing 12% oxygen (hypoxia) under photopic illumination. The hypoxia visual field data were compared with the field data measured during normoxia. Absolute sensitivities (in dB) were analysed in seven concentric rings at 1°, 3°, 6°, 10°, 15°, 22° and 30° eccentricities as well as mean defect (MD) and pattern defect (PD) were calculated. Preliminary data are reported for mesopic light levels. Results: Under photopic illumination, flicker and static visual field sensitivities at all eccentricities were not significantly different between hypoxia and normoxia conditions. The mean defect and pattern defect were not significantly different for either test between the two oxygenation conditions. Conclusion: Although flicker stimulation increases cellular metabolism, flicker photopic visual field impairment is not detected during mild hypoxia. These findings contrast with electrophysiological flicker tests in young participants that show impairment at photopic illumination during the same levels of mild hypoxia. Potential mechanisms contributing to the difference between the visual fields and electrophysiological flicker tests including variability in perimetric data, neuronal adaptation and vascular autoregulation, are considered. The data have implications for the use of visual perimetry in the detection of ischaemic/hypoxic retinal disorders under photopic and mesopic light levels.

Dextran and protamine-based solid lipid nanoparticles as potential vectors for the treatment of X-linked juvenile retinoschisis

This is a copy of an article published in the Human gene therapy © 2012 copyright Mary Ann Liebert, Inc.; Human gene therapy is available online at: http://online.liebertpub.com.

Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice

Fundus autofluorescence (AF) imaging by confocal scanning laser ophthalmoscopy has been widely used by ophthalmologists in the diagnosis/monitoring of various retinal disorders. It is believed that fundus AF is derived from lipofuscin in retinal pigment epithelial (RPE) cells; however, direct clinicopathological correlation has not been possible in humans. We examined fundus AF by confocal scanning laser ophthalmoscopy and confocal microscopy in normal C57BL/6 mice of different ages. Increasingly strong AF signals were observed with age in the neuroretina and subretinal/RPE layer by confocal scanning laser ophthalmoscopy. Unlike fundus AF detected in normal human subjects, mouse fundus AF appeared as discrete foci distributed throughout the retina. Most of the AF signals in the neuroretina were distributed around retinal vessels. Confocal microscopy of retinal and choroid/RPE flat mounts demonstrated that most of the AF signals were derived from Iba-1+ perivascular and subretinal microglia. An age-dependent accumulation of Iba-1+ microglia at the subretinal space was observed. Lipofuscin granules were detected in large numbers in subretinal microglia by electron microscopy. The number of AF+ microglia and the amount of AF granules/cell increased with age. AF granules/lipofuscin were also observed in RPE cells in mice older than 12 months, but the number of AF+ RPE cells was very low (1.48 mm-2 and 5.02 mm-2 for 12 and 24 months, respectively) compared to the number of AF+ microglial cells (20.63 mm-2 and 76.36 mm-2 for 6 and 24 months, respectively). The fluorescence emission fingerprints of AF granules in subretinal microglia were the same as those in RPE cells. Our observation suggests that perivascular and subretinal microglia are the main cells producing lipofuscin in normal aged mouse retina and are responsible for in vivo fundus AF. Microglia may play an important role in retinal aging and age-related retinal diseases.

Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management.

From gene to protein: Investigating the disease etiology of retinitis pigmentosa

Retinitis pigmentosa (RP) is a name given to a group of inherited retinal dystrophies that lead to progressive photoreceptor degeneration, and thus, visual impairment. It is evident at both the clinical and the molecular level that these are heterogeneous disorders, with wide variation in severity, mode of inheritance, and phenotype. The genetics of RP are not simple; the disease can be inherited in dominant, recessive, X-linked, and digenic modes. Autosomal dominant RP (adRP) results from mutations in at least ten mapped loci, but there may be dozens of genetic loci where mutations can cause RP. To date, there are over a hundred genes known to cause retinal degenerative diseases, and less than half of these have been cloned (RetNet). Among the dozens of retinitis pigmentosa loci known to exist, only a few have been identified and the remainders are inferred from linkage studies. Today, the genes for seven of the twelve-adRP loci have been identified, and these are rhodopsin, peripherin/RDS, NRL, ROM1, CRX, RP13 and RP1. My research projects involved a combination of the continued search for genes involved in retinal dystrophies, as well the investigation into the role of peripherin/RDS and RP1 in the disease etiology of autosomal dominant RP. ^ Most of the mutations leading to inherited retinal disorders have been identified in predominately retina expressed genes like rhodopsin, peripherin/RDS, and RP1. Expressed sequence tags (ESTs) that were retina-specific were culled from sequence databases and, together with laboratory analysis, were analyzed as potential candidate genes for retinal dystrophies. Thirteen of the fifty-five identified retina-specific ESTs mapped to within candidate regions for inherited retinopathies. One of these is RP1L1, a homologue of RP1 and a potential cause of adRP. ^ Once a disease-associated gene has been identified, elucidating the role of that gene in the visual process is essential for understanding what happens when the process is defective as it is in adRP. My next projects involved investigating the role of a novel 5′ donor +3 splice site mutation on the mRNA of peripherin/RDS in adRP affected individuals, and comparative sequencing in RP1 to define conserved regions of the protein. Comparative sequencing is a powerful way to delineate critical regions of a sequence because different regions of a gene have different functions, and each region is subject to different levels of functional or structural constraints. Establishing a framework of conserved domains is beneficial not only for structural or functional studies, but can also aid in determining the potential effects of mutations. With the completion of sequencing of human genome, and other organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans , and Drosophila, the facility of comparative sequencing will only increase in the future. Comparative sequencing has already become an established procedure for pinpointing conserved regions of a protein, and is an efficient way to target regions of a protein for experimental and/or evolutionary analysis. ^

An overview of eye-related photophobias

This chapter provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is the experience of discomfort affecting the eyes as a result of exposure to light. It has a variety of causes, including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for photophobia associated with these conditions: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Eye-related photophobia

This article provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is an experience of discomfort affecting the eyes due to exposure to light. It has a variety of causes including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for eye-related photophobia: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18–30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.

Dynamic, invivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress