Role of glast-positive glial cells during photoreceptor development

Purpose: Taking advantage of two transgenic lines, glast.DsRed and crx.gfp, that express fluorescent proteins in glial and photoreceptor cells respectively, we investigate the role of glast-positive glial cells (GPCs) in the survival/differentiation/proliferation of age-matched photoreceptor cells. Methods: Primary retinal cells were isolated from newborn transgenic mouse retina (glast.dsRed::crx.gfp) at postnatal day (P0/P1) and propagated in defined medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (bFGF). By flow-sorting another population of pure GPCs was isolated. Both populations were expanded and analyzed for the presence of specific retinal cell markers. Notably, the primary cell culture collected from the transgenic line glast.dsRed::crx.gfp showed a conspicuous presence of immature photoreceptors growing on top of GPCs. In order to reveal the role of such cells in the survival/differentiation/proliferation of photoreceptors we set up in vitro cultures of retina-derived cells that allowed long-term time-lapse recordings charting every cell division, death and differentiation event. To assess the regenerative potential of GPCs we challenged them with compounds mimicking retinal degeneration (NMU, NMDA, Zaprinast). Mass spectrometry (MS), immunostainings and other molecular approaches were performed to reveal adhesion molecules involved in the relationship between glial cells and photoreceptors. Results: Both primary cell lines were highly homogenous, with an elongated morphology and the majority expressed Müller glia markers (MG) such as glast, blbp, glt-1, vimentin, glutamine synthetase (GS), GFAP, cd44, mash1 and markers of reactive Müller glia such as nestin, pax6. Conversely, none of them were found positive for retinal neuron markers like tuj1, otx2, recoverin. Primary cultures of GPCs show the incapability of glial cells to give rise to photoreceptors in both wild type or degenerative environment. Furthermore, primary cultures of pure GPCs challenged with different compounds did not highlight the production of new glial cell-derived photoreceptors. Adhesion molecules involved in the contact between photoreceptors and glial cells are still under investigation. Conclusions: Primary glia cells do not give rise to photoreceptor cells in wt and degenerative conditions at least in vitro. The roles of glial cells seem to be more linked to the maintenance/proliferation of photoreceptor cells.

Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

A Novel Homozygous RPE65 Mutation in Leber Congenital Amaurosis Associated With Cone-Rod Dystrophy

Purpose: To report the clinical and genetic study of a family with Leber congenital amaurosis (LCA). Methods: We studied a consanguineous family from Yemen in which three individuals were affected with LCA. Genomic DNA was prepared from venous leukocytes. Linkage analysis of all family members using polymorphic markers flanking the known LCA genes was performed, followed by direct sequencing of all the exons and intron-exon junctions of the RPE65 gene. Results: The three affected were 5, 8 and 12 years old. Severe visual impairment and night blindness were noticed during infancy. Nystagmus was not a feature. Photophobia was only observed in the 8-year-old patient. The 5-year old youngest affected had a bilateral hyperopia of +3.50 and a visual acuity of 1/60. The oldest two had mild myopia and visual acuity limited to hand movements RE and counting fingers LE for the oldest and of 5/60 OD, 6/60 OS for the other. On fundus examination, they harbored common clinical features such as disc pallor, attenuated vessels, white flecks in the retina mid-periphery and bull's eye maculopathy. Electroretinograms of the oldest child were completely extinguished while residual scotopic responses with abolished photopic and flicker responses were observed in the two youngest. Sequencing identified a novel missense mutation, IVS2-3C>G, in the second RPE65 intron. The mutation was not detected in 80 ethnically matched normal individuals. Conclusion: We have identified a novel LCA-related homozygous RPE65 mutation associated with a severe clinical presentation including an early and severe cone dysfunction. This is in contrast with the presentation associated with other RPE65 mutations predominantly causing a rod-cone dystrophy with residual cone function. The identified mutation potentially affects splicing of the third exon and could result in a loss of function. Definite functional consequences of this change still need to be characterized.

Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.

Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease.

Double blind controlled trial of D-penicillamine in patients with primary biliary cirrhosis.

One hundred and eighty nine patients with primary biliary cirrhosis were entered into a double blind, placebo controlled randomised trial starting in January 1978 to assess the therapeutic value of d-penicillamine 1200 mg daily. Eighteen of the 98 patients receiving d-penicillamine and 22 of the 91 placebo treated patients died during the study. Thirty six per cent of those on d-penicillamine and 8% of those on placebo were withdrawn from the study. No difference in overall survival was noted between the two groups of patients whether the results were analysed for the entire period of observation or only during the period in which the patients were receiving therapy. The mortality rate of those receiving d-penicillamine in histological stage I to II, however, was one third of that of the placebo group although this difference did not reach statistical significance. Using the occurrence rate ratio as the statistical method of analysis, no effect of d-penicillamine was noted on any clinical, biochemical or hist

A comparison of the accuracy of peritoneoscopy and liver biopsy in the diagnosis of cirrhosis

The accuracy of peritoneoscopy and liver biopsy in the diagnosis of hepatic cirrhosis was compared in 473 consecutive patients submitted to both procedures. One hundred and fifty-two of them had cirrhosis diagnosed by one or both methods. There was 73% agreement between the two procedures. `Apparent' false-negative results were 17·7% for peritoneoscopy and 9·3% for liver biopsy. The incidence of false-negative results in the diagnosis of cirrhosis can be reduced by combining both procedures.

Upper gastrointestinal bleeding in cirrhosis: clinical and endoscopic correlations

The clinical data of 180 episodes of upper gastrointestinal bleeding in 168 patients with cirrhosis of the liver are examined. The source of bleeding had been determined by early endoscopy in all cases. In men under the age of 50 years, and without symptoms of liver failure, bleeding was due to ruptured gastro-oesophageal varices in 84% of cases. Severe liver failure was associated with acute lesions of gastric mucosa in many cases. No presumptive diagnosis of the source of haemorrhage could be based on the examination of other clinical data (presence of ascites, mode of presentation and pattern of bleeding, history of ulcer disease, alcoholism, and previous medication.

Role of the c-Jun N-terminal kinase pathway in retinal excitotoxicity, and neuroprotection by its inhibition.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06705.x Abstract Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c-Jun N-terminal kinase (JNK) activation in NMDA-mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, d-form of JNK-inhibitor 1 (d-JNKI-1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell-permeable peptide d-JNKI-1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC-labeled d-JNKI-1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA-induced phosphorylation of JNK. Injections of unlabeled d-JNKI-1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA-induced calpain-specific fodrin cleavage was likewise strongly inhibited by d-JNKI-1. Moreover the electroretinogram was partially preserved by d-JNKI-1. Thus, the JNK pathway is involved in NMDA-mediated retinal excitotoxicity and JNK inhibition by d-JNKI-1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.

Mineralocorticoid Receptor Antagonism in the Treatment of Chronic Central Serous Chorioretinopathy : A Pilot Study.

PURPOSE:: Based on experimental data showing that central serous chorioretinopathy could result from overactivation of mineralocorticoid receptor pathway in choroid vessels, the authors studied eplerenone, a mineralocorticoid receptor antagonist, as a potential treatment for chronic central serous chorioretinopathy. METHODS:: This nonrandomized pilot study included 13 patients with central serous chorioretinopathy of at least 4-month duration, treated with 25 mg/day of oral eplerenone for a week followed by 50 mg/day for 1 or 3 months. The primary outcome measure was the changes in central macular thickness recorded by optical coherence tomography, and the secondary outcomes included changes in foveal subretinal fluid (SRF) measured by OCT, in best-corrected visual acuity (BCVA) and the percentage of eyes achieving complete resolution of subretinal fluid during the treatment period. RESULTS:: Central macular thickness decreased significantly from 352 ± 139 μm at baseline to 246 ± 113 μm and 189 ± 99 μm at 1 and 3 months under eplerenone treatment (P < 0.05 and P < 0.01, respectively). At 3 months, the subretinal fluid significantly decreased compared with baseline subretinal fluid (P < 0.01) and best-corrected visual acuity significantly improved compared with baseline best-corrected visual acuity (P < 0.001). CONCLUSION:: Eplerenone treatment was associated with a significant reduction in central macular thickness, subretinal fluid level, and an improvement in visual acuity. Randomized controlled trials are needed to confirm these encouraging results.

Upper gastrointestinal bleeding in cirrhosis: clinical and endoscopic correlations

The clinical data of 180 episodes of upper gastrointestinal bleeding in 168 patients with cirrhosis of the liver are examined. The source of bleeding had been determined by early endoscopy in all cases. In men under the age of 50 years, and without symptoms of liver failure, bleeding was due to ruptured gastro-oesophageal varices in 84% of cases. Severe liver failure was associated with acute lesions of gastric mucosa in many cases. No presumptive diagnosis of the source of haemorrhage could be based on the examination of other clinical data (presence of ascites, mode of presentation and pattern of bleeding, history of ulcer disease, alcoholism, and previous medication.

Etude de Nkx5-3, un facteur de transcription impliqué dans le développement de l'oeil

Résumé :Une famille souffrant d'un nouveau syndrome oculo-auriculaire, appelé syndrome de Schorderet-Munier, a été identifiée. Ce syndrome est caractérisé par une déformation du lobe de l'oreille et des anomalies ophtalmiques, notamment une microphtalmie, une cataracte, un colobome et une dégénérescence rétinienne. Le gène impliqué dans ce syndrome est NKX5-3 codant un facteur de transcription contenant un homéodomaine. Chez les patient atteints, le gène comporte une délétion de 26 nucléotides provoquant probablement l'apparition d'un codon stop précoce. Ce gène n'est exprimé que dans certains organes dont les testicules et les ganglions cervicaux supérieurs, ainsi que dans les organes touchés par ce syndrome, à savoir le pavillon de l'oreille et l'oeil, surtout lors du développement embryonnaire. Au niveau de la rétine, NKX5-3 est présent dans la couche nucléaire interne et dans la couche dè cellules ganglionnaires et est exprimé de manière polarisée selon un axe temporal > nasal et ventral > dorsal. Son expression in vitro est régulée par Spl, un facteur de transcription exprimé durant le développement de l'oeil chez la souris. NKX5-3 semble lui-même provoquer une inhibition de l'expression de SHH et de EPHA6. Ces gènes sont tous les deux impliqués à leur manière dans le guidage des axones des cellules ganglionnaires de la rétine. Pris ensemble, ces résultats nous permettent donc d'émettre une hypothèse quant à un rôle potentiel de NKX5-3 dans ce processus.Abstract :A family with a new oculo-auricular syndrome, called syndrome of Schorderet-Munier, was identified. This disease is characterised by a deformation of the ear lobule and by several ophthalmic abnormalities, like microphthalmia, cataract, coloboma and a retinal degeneration. The gene, which causes this syndrome, is NKX5-3 coding for a transcription factor contaning a homeodomain. In the affectd patients, the defect consists of a deletion of 26 nucleotides probably producing a premature stop codon. This gene is only expressed in a few organs like testis and superior cervical ganglions, as well as in organs affected by this syndrome, namely the ear pinna and the eye, mainly during embryonic development. In the retina, NKX5-3 is present in the inner nuclear layer and in the ganglion cells layer. It is expressed along a gradient ranging from the temporal retina to nasal retina and from the ventral to the dorsal part. Its in vitro expression is regulated by Spl, a transcription factor expressed during the murine eye development. NKX5-3 seems to inhibit the expression of SHH and EPHA6. These genes are both implicated, in their own way, in the axon guidance of the retinal ganglion cells. Taken together, these results allow us to make an assumption about a potential role of NKX5-3 in this process.

Enhanced oligonucleotide delivery to mouse retinal cells using iontophoresis.

PURPOSE: To study the combination of oligodeoxynucleotides (ODNs) intravitreous injection and saline transpalpebral iontophoresis on the delivery of ODNs to photoreceptors in the newborn rd1/rd1 mice. METHODS: Cathodal or anodal transpalpebral iontophoresis (1.43 mA/cm(2) for 5 min) was applied to eyes of postnatal day 7 (PN7) rd1/rd1 mice immediately before the intravitreous injection of ODNs. The effect of cathodal iontophoresis after ODNs injection was also evaluated. The influence of current intensity (0.5, 1.5, and 2.5 mA) was assayed with cathodal iontophoresis performed prior to ODNs injection. The duration of current-induced facilitation of ODNs delivery to photoreceptors was evaluated for 6 h following iontophoresis. One group of control eyes received cathodal iontophoresis prior to the intravitreous injection of phosphate buffered saline (PBS) or hexachlorofluorescein (Hex). The second control group received ODN or Hex intravitreous injection without iontophoresis. The penetration of fluorescent ODNs in the outer nuclear layer (ONL) was quantified by image analysis of the ONL fluorescence intensity on cryosection microphotographs. Integrity of ODN was assessed using acrylamide gel migration after its extraction from the retina of treated mice. The integrity of retinal structure, 1 and 24 h after iontophoresis, was analyzed using light and electron microscopy. RESULTS: Transpalpebral anodal or cathodal saline iontophoresis enhanced the penetration of ODNs in all retinal layers. Cathodal iontophoresis was more efficient than anodal iontophoresis in enhancing the tissue penetration of the injected ODN. Photoreceptor delivery of ODN was significantly higher when cathodal saline transpalpebral iontophoresis was applied prior than after the injection. The extent of enhanced tissue penetration decreased in parallel to the increased interval between iontophoresis application and the intravitreous injection. Current of 1.5 mA was safe and optimal for the delivery of ODNs to the ONL. One hour after iontophoresis followed by injection, ODN extracted from the retina of treated eyes remained intact. Histology and electron microscopy observations demonstrated that iontophoresis using the optimal parameters did not induce any permanent tissue alterations or structure damage. CONCLUSIONS: Saline transpalpebral iontophoresis facilitates the penetration of injected ODNs in photoreceptors for at least 3 h. This method may be considered for photoreceptor targeted gene therapy.

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for idiopathic macular epiretinal membrane.

PURPOSE: To evaluate the histologic features of cellular retinal fragments on the internal limiting membrane (ILM) removed during idiopathic macular epiretinal membrane (MEM) peeling surgery with and without the aid of indocyanine green (ICG) diluted in 5% glucose. METHODS: ILM specimens removed from 88 eyes during idiopathic MEM surgery between 1995 and 2003 were reviewed retrospectively. Histologic analysis focused on the presence and characteristics of retinal fragments on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery) and group II (ICG-assisted peeling). RESULTS: Seventy-one eyes underwent MEM surgery without the aid of ICG (group I) and seventeen underwent MEM ICG-assisted surgery (group II). The amount of Müller cell debris on the retinal surface of the ILM was more significant in the group I than in the group II (40.8 vs. 11.8; P = 0.024). Large fragments of Müller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4 vs. 23.5%; P = 0.003). CONCLUSIONS: The use of ICG diluted with 5% glucose in ILM removal during MEM surgery was associated with less retinal debris attached to the retinal face of the ILM compared with surgery in which ICG was not used.