Novel Cone Transducin Alpha Subunit Mutation In Tunisian Patients And Genotype-phenotype Correlation In Complete Achromatopsia

Purpose: Complete achromatopsia is a rare autosomal recessive disease due to CNGA3, CNGB3, GNAT2 and PDE6C mutations. We studied a large consanguineous Tunisian family including twelve individuals.Methods: Ophthalmic evaluation included a full clinical examination, color vision testing, optical coherence tomography and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing.Results: In all affected subjects, acuity ranged from 20/50 to 20/200. Fundus examination was normal except for two patients who had respectively 4 mm and 5 mm diameters of peripheral congenital hypertrophy. Likewise retinal layers exploration by OCT revealed no change in the thickness of the central retina. Color Vision with 100 Hue Farnsworth test described a profound color impairment along all three axes of color vision. The haplotype analysis of GNAT2 markers revealed that all affected offspring were homozygous by descent for the four polymorphic markers. The maximum lod score value, 4.33, confirmed the evidence for linkage to the GNAT2 gene.A homozygous novel nonsense mutation R313X was identified segregating with an identical GNAT2 haplotype in all affected subjects. This mutation could interrupt interaction with photoactivated rhodopsin, resulting in a failure of visual transduction. In fact, ERG showed a clearly abolished photopic b-wave and flicker responses with no residual cone function justifying the severe GNAT2 achromatopsia phenotype.Conclusions: This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and of the largest family with recessive achromatopsia involving GNAT2, thus providing a unique opportunity for genotype phenotype correlation for this extremely rare condition.

Optimization Of Promoter Sequences In Order To Mimic Physiological Pde6b Expression

Purpose: Many retinal degenerations result from defective retina-specific gene expressions. Thus, it is important to understand how the expression of a photoreceptor-specific gene is regulated in vivo in order to achieve successful gene therapy. The present study aims to design an AAV2/8 vector that can regulate the transcript level in a physiological manner to replace missing PDE6b in Rd1 and Rd10 mice. In previous studies (Ogieta, et al., 2000), the short 5' flanking sequence of the human PDE6b gene (350 bp) was shown to be photoreceptor-specific in transgenic mice. However, the efficiency and specificity of the 5' flanking region of the human PDE6b was not investigated in the context of gene therapy during retinal degeneration. In this study, two different sequences of the 5' flanking region of the human PDE6b gene were studied as promoter elements and their expression will be tested in wild type and diseased retinas (Rd 10 mice).Methods: Two 5' flanking fragments of the human PDE6b gene: (-93 to +53 (150 bp) and -297 to +53 (350 bp)) were cloned in different plasmids in order to check their expression in vitro and in vivo by constructing an AAV2/8 vector. These elements drove the activity of either luciferase (pGL3 plasmids) or EGFP. jetPEI transfection in Y 79 cells was used to evaluate gene expression through luciferase activity. Constructs encoding EGFP under the control of the two promoters were performed in AAV2.1-93 (or 297)-EGFP plasmids to produce AAV2/8 vectors.Results: When pGL3-93 (150 bp) or pGL3-297 (350 bp) were transfected in the Y-79 cells, the smaller fragment (150 bp) showed higher gene expression compared to the 350 bp element and to the SV40 control, as previously reported. The 350 bp drove similar levels of expression when compared to the SV40 promoter. In view of these results, the fragments (150 bp or 350 bp) were integrated into the AAV2.1-EGFP plasmid to produce AAV2/8 vector, and we are currently evaluating the efficiency and specificity of the produced constructs in vivo in normal and diseased retinas.Conclusions: Comparisons of these vectors with vectors bearing ubiquitous promoters should reveal which construct is the most suitable to drive efficient and specific gene expression in diseased retinas in order to restore a normal function on the long term.

Light-emitting diodes (LED) for domestic lighting: any risks for the eye?

Light-emitting diodes (LEDs) are taking an increasing place in the market of domestic lighting because they produce light with low energy consumption. In the EU, by 2016, no traditional incandescent light sources will be available and LEDs may become the major domestic light sources. Due to specific spectral and energetic characteristics of white LEDs as compared to other domestic light sources, some concerns have been raised regarding their safety for human health and particularly potential harmful risks for the eye. To conduct a health risk assessment on systems using LEDs, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES), a public body reporting to the French Ministers for ecology, for health and for employment, has organized a task group. This group consisted physicists, lighting and metrology specialists, retinal biologist and ophthalmologist who have worked together for a year. Part of this work has comprised the evaluation of group risks of different white LEDs commercialized on the French market, according to the standards and found that some of these lights belonged to the group risk 1 or 2. This paper gives a comprehensive analysis of the potential risks of white LEDs, taking into account pre-clinical knowledge as well as epidemiologic studies and reports the French Agency's recommendations to avoid potential retinal hazards.

Retinal vessel oxygen saturation and its correlation with structural changes in retinitis pigmentosa.

PURPOSE: To study the influence of retinal structural changes on oxygen saturation in retinitis pigmentosa (RP) patients. METHODS: Oximetry measurements were performed on 21 eyes of 11 RP patients and compared to 24 eyes of 12 controls. Retinal oxygen saturation was measured in all major retinal arterioles (A-SO₂) and venules (V-SO₂) with an oximetry unit of the retinal vessel analyser (IMEDOS Systems UG, Jena, Germany). Oximetry data were compared with morphological changes measured by Cirrus optical coherence tomography (OCT) (Carl Zeiss Meditec, Dublin, CA, USA, macular thickness protocol). RESULTS: In RP patients, the retinal A-SO₂ and V-SO₂ levels were higher at 99.3% (p = 0.001, anova based on mixed-effects model) and 66.8% (p < 0.001), respectively, and the difference between the two (A-V SO₂) was lower at 32.5% (p < 0.001), when compared to the control group (92.4%; 54.0%; 38.4%, respectively). With the RP group, the A-V SO₂ correlated positively, not only with central macular thickness, but also with retinal thickness, in zones 2 and 3 (p = 0.006, p = 0.007, p = 0.014). CONCLUSION: These data indicate that oxygen metabolism was altered in RP patients. Based on our preliminary results, retinal vessel saturation correlated with structural alterations in RP. This method could be valuable in monitoring disease progression and evaluating a potential therapeutic response.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes.

PURPOSE: To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS: Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS: When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS: Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

Topical and intravitreous administration of cationic nanoemulsions to deliver antisense oligonucleotides directed towards VEGF KDR receptors to the eye.

Antisense oligonucleotides (ODNs) specific for VEGFR-2-(17 MER) and inhibiting HUVEC proliferation in-vitro were screened. One efficient sequence was selected and incorporated in different types of nanoemulsions the potential toxicity of which was evaluated on HUVEC and ARPE19 cells. Our results showed that below 10 microl/ml, a 2.5% mid-chain triglycerides cationic DOTAP nanoemulsion was non-toxic on HUVEC and retinal cells. This formulation was therefore chosen for further experiments. In-vitro transfection of FITC ODNs in ARPE cells using DOTAP nanoemulsions showed that nanodroplets do penetrate into the cells. Furthermore, ODNs are released from the nanoemulsion after 48 h and accumulate into the cell nuclei. In both ex-vivo and in-vivo ODN stability experiments in rabbit vitreous, it was noted that the nanoemulsion protected at least partially the ODN from degradation over 72 h. The kinetic results of fluorescent ODN (Hex) distribution in DOTAP nanoemulsion following intravitreal injection in the rat showed that the nanoemulsion penetrates all retinal cells. Pharmacokinetic and ocular tissue distribution of radioactive ODN following intravitreal injection in rabbits showed that the DOTAP nanoemulsion apparently enhanced the intraretinal penetration of the ODNs up to the inner nuclear layer (INL) and might yield potential therapeutic levels of ODN in the retina over 72 h post injection.

Surgical treatment of lamellar macular hole associated with epimacular membrane.

BACKGROUND:: Although the surgical treatment of full-thickness macular hole is well established, the utility of pars plana vitrectomy in the treatment of lamellar macular hole (LMH) remains less clear. The purpose of the study is to report functional results of surgical treatment of LMH associated with epiretinal membrane. METHODS:: Retrospective chart review of patients undergoing pars plana vitrectomy and peeling of epiretinal membrane and internal limiting membrane, with or without air or gas tamponade, for symptomatic LMH associated with epimacular membrane. RESULTS:: Forty-five eyes of 44 patients were operated for LMH associated with epimacular membrane between May 2000 and July 2009. Pars plana vitrectomy and membrane peeling were combined with air or gas tamponade in 43 of 45 cases. Mean logarithm of the minimum angle of resolution best-corrected visual acuity improved from 0.4 preoperatively to 0.13 postoperatively (P < 0.0001). Improvement in visual acuity ranged from 0 Early Treatment Diabetic Retinopathy Study (ETDRS) lines to 8.9 ETDRS lines (mean, 2.65 ETDRS lines). Visual acuity improved by ≥1 ETDRS line(s) in 40 of 45 eyes (89%) and by ≥2 ETDRS lines in 26 of 45 eyes (58%) after the surgical procedure. No patient lost vision. CONCLUSION:: This small retrospective study suggests that surgical treatment of LMH associated with epimacular membrane may improve visual acuity in symptomatic patients.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 μg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Clinical and electrophysiological recovery in Leber hereditary optic neuropathy with G3460A mutation.

To report a case of clinical and electrophysiological recovery in Leber hereditary optic neuropathy (LHON) with G3460A Mutation. A 10-year-old boy with a three-month history of painless bilateral sequential visual loss upon presentation underwent visual acuity (diminished), anterior and posterior segment examination (normal), fluorescein angiography (normal), Goldman kinetic perimetry (bilateral central scotomata), genetic (a point G3460A mutation) and electrophysiological investigation (undetectable pattern visual evoked potentials (VEP); low amplitude, broadened and reduced flash VEPs and loss of the N95 component in the pattern electroretinograms). Diagnosis of LHON was made. Eighteen months later vision and electrophysiological tests results began spontaneously improving. Kinetic perimetry revealed reduced density and size of scotomata. Two years later, there had been further electrophysiological improvement. This report describes both clinical and electrophysiological improvement in LHON with G3460A mutation.

A system-level, molecular evolutionary analysis of mammalian phototransduction

Visual perception is initiated in the photoreceptor cells of the retina via the phototransduction system.This system has shown marked evolution during mammalian divergence in such complex attributes as activation time and recovery time. We have performed a molecular evolutionary analysis of proteins involved in mammalianphototransduction in order to unravel how the action of natural selection has been distributed throughout thesystem to evolve such traits. We found selective pressures to be non-randomly distributed according to both a simple protein classification scheme and a protein-interaction network representation of the signaling pathway. Proteins which are topologically central in the signaling pathway, such as the G proteins, as well as retinoid cycle chaperones and proteins involved in photoreceptor cell-type determination, were found to be more constrained in their evolution. Proteins peripheral to the pathway, such as ion channels and exchangers, as well as the retinoid cycle enzymes, have experienced a relaxation of selective pressures. Furthermore, signals of positive selection were detected in two genes: the short-wave (blue) opsin (OPN1SW) in hominids and the rod-specific Na+/Ca2+,K+ ion exchanger (SLC24A1) in rodents. The functions of the proteins involved in phototransduction and the topology of the interactions between them have imposed non-random constraints on their evolution. Thus, in shaping or conserving system-level phototransduction traits, natural selection has targeted the underlying proteins in a concerted manner.