Susceptibility of streptozotocin-induced diabetic rat retinal function and ocular blood flow to acute intraocular pressure challenge.

PURPOSE: To consider whether STZ-induced hyperglycemia renders rat retinal function and ocular blood flow more susceptible to acute intraocular pressure (IOP) challenge.

METHODS: Retinal function (electroretinogram, ERG) was measured during acute IOP challenge (10-100 mmHg, 5 mmHg increments, 3 min/step, vitreal cannulation) in adult Long-Evans rats (6-week old, citrate: n=6, STZ: n=10) 4 weeks after citrate buffer or streptozotocin (STZ, 65 mg/kg, blood glucose > 15 mmol/l) injection. At each IOP, dim and bright flash (-4.56, -1.72 log cd.s.m^-2) ERG responses were recorded to measure inner retinal and ON-bipolar cell function, respectively. Ocular blood flow (laser Doppler flowmetry, citrate; n=6, STZ; n=10) was also measured during acute IOP challenge. Retinae were isolated for qPCR analysis of nitric oxide synthase mRNA expression endothelial, eNos; inducible, iNos; neuronal, nNos).

RESULTS: STZ-induced diabetes increased the susceptibility of inner retinal (IOP at 50% response, 60.1, CI: 57.0-62.0 mmHg vs. citrate: 67.5, CI: 62.1-72.4 mmHg) and ON-bipolar cell function (STZ: 60.3, CI: 58.0-62.8 mmHg vs. citrate: 65.1, CI: 58.0-62.78 mmHg) and ocular blood flow (43.9, CI: 40.8-46.8 vs. citrate: 53.4, CI: 50.7-56.1 mmHg) to IOP challenge. Citrate eyes showed elevated eNos mRNA (+49.7%) after IOP stress, an effect not found in STZ-diabetic eyes (-5.7%, P<0.03). No difference was observed for iNos or nNos (P>0.05) following IOP elevation.

CONCLUSIONS: STZ-induced diabetes increased functional susceptibility during acute IOP challenge. This functional vulnerability is associated with a reduced capacity for diabetic eyes to upregulate eNOS expression and to autoregulate blood flow in response to stress.

Extremely Complex Populations of Small RNAs in the Mouse Retina and RPE/Choroid

Purpose: MicroRNAs (miRNAs) are small non-coding RNAs of ~18-22 nucleotides in length that regulate gene expression. They are widely expressed in the retina, being both required for its normal development and perturbed in disease. The aim of this study was to apply new high-throughput sequencing techniques to more fully characterise the microRNAs and other small RNAs expressed in the retina and retinal pigment epithelium (RPE)/choroid of the mouse.

Methods: Retina and RPE/choroid were dissected from eyes of 3 month-old C57BL/6J mice. Small RNA libraries were prepared and deep sequencing performed on a Genome Analyzer (Illumina). Reads were annotated by alignment to miRBase, other non-coding RNA databases and the mouse genome.

Results: Annotation of 9 million reads to 320 microRNAs in retina and 340 in RPE/choroid provides the most comprehensive profiling of microRNAs to date. Two novel microRNAs were identified in retina. Members of the sensory organ specific miR-183,-182,-96 cluster were amongst the most highly expressed, retina-enriched microRNAs. Remarkably, microRNA 'isomiRs', which vary slightly in length and are differentially detected by Taqman RT-PCR assays, existed for all the microRNAs identified in both tissues. More variation occurred at the 3' ends, including non-templated additions of T and A. Drosha-independent mirtron microRNAs and other small RNAs derived from snoRNAs were also detected.

Conclusions: Deep sequencing has revealed the complexity of small RNA expression in the mouse retina and RPE/choroid. This knowledge will improve the design and interpretation of future functional studies of the role of microRNAs and other small RNAs in retinal disease.

A Longitudinal Study of Stargardt Disease: Quantitative Assessment of Fundus Autofluorescence, Progression and Genotype Correlations

PURPOSE To characterise subtypes of fundus autofluorescence (AF), the progression of retinal atrophy and correlate these findings with genotype in Stargardt Disease. METHODS Full clinical examination and AF imaging was undertaken in 68 patients with Stargardt Disease. The baseline data were compared with those at follow-up. Patients were classified into three AF subtypes: type 1 had a localised low signal at the fovea surrounded by a homogeneous background; type 2 had a localised low signal at the macula surrounded by a heterogeneous background with numerous foci of abnormal signal; type 3 had multiple low signal areas at the posterior pole with a heterogeneous background. At baseline, there were 19 patients with type 1, 41 with type 2, and 8 with type 3. The areas of reduced AF signal were measured and rate of atrophy enlargement (RAE) was calculated as the difference of the atrophy size over time (mm2) divided by the follow-up interval (yrs). Molecular screening of ABCA4 was undertaken. RESULTS The mean follow-up interval was 9.1 years. 42% of type 1 progressed to type 2, and 12% of type 2 progressed to type 3. RAE (mm2/yr) based upon baseline AF subtypes was significantly different; 0.06 in type 1, 0.67 in type 2, and 4.37 in type 3. ABCA4 variants were identified in 57 patients. There was a significant association between AF subtype and genotype. CONCLUSIONS The AF pattern at baseline influences the enlargement of atrophy over time and has genetic correlates. These data are likely to assist in the provision of counselling on prognosis in Stargardt Disease and be valuable for future clinical trials.

The Role of K+ and Cl- Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow.

Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1).

Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10µl) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1µM) and iberiotoxin (4µM), and the IKv-inhibitor correolide (40µM) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide.

Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

GABAergic control of arteriolar diameter in the rat retina

Purpose: To investigate the role of γ-aminobutryic acid (GABA) in the regulation of arteriolar diameter in the rat retina.

Methods.: The actions of GABA on arteriolar diameter were examined using ex vivo retinal whole-mount preparations and isolated vessel segments. In most experiments, arterioles were partially preconstricted with endothelin (Et)-1. The expression levels of GABAA and GABAB receptors on isolated rat retinal Müller cells were assessed by immunohistochemistry.

Results.: GABA (0.1–1 mM) evoked vasodilation or vasoconstriction of arterioles in whole-mount preparations. No such effects were observed with isolated vessel segments. In whole mount samples, the GABAA receptor agonist muscimol caused vasomotor responses in only a small proportion of vessels. In contrast, arteriolar responses to the GABAB receptor agonists baclofen and SKF97541 more closely resembled those observed with GABA. No responses were seen with the GABAC receptor agonist 5-methylimidazoleacetic acid. GABA-induced vasodilator responses were, for the most part, repeatable in the presence of the GABAA receptor antagonist bicuculline. These responses, however, were completely blocked in the presence of the GABAB receptor inhibitor 2-hydroxysaclofen. Strong immunolabeling for both GABAA and GABAB receptors was detected in isolated Müller cells. In the absence of Et-1–induced preconstriction, most vessels were unresponsive to bicuculline or 2-hydroxysaclofen.

Conclusions.: GABA exerts complex effects on arteriolar diameter in the rat retina. These actions appear largely dependent upon the activation of GABAB receptors in the retinal neuropile, possibly those located on perivascular Müller cells. Despite these findings, endogenous GABA appears to contribute little to the regulation of basal arteriolar diameter in the rat retina.

Suggestive association with ocular phoria at chromosome 6p22

PURPOSE. We conducted a genome-wide association study to identify genetic factors that contribute to the etiology of heterophoria.

METHODS. We measured near and far vertical and horizontal phorias in 988 healthy adults aged 16 to 40 using the Keystone telebinocular with plates 5218 and 5219. We regressed degree of phoria against genotype at 642758 genetic loci. To control for false positives, we applied the conservative genome-wide permutation test to our data.

RESULTS. A locus at 6p22.2 was found to be associated with the degree of near horizontal phoria (P = 2.3 × 10 ). The P value resulting from a genome-wide permutation test was 0.014.

CONCLUSIONS. The strongest association signal arose from an intronic region of the gene ALDH5A1, which encodes the mitochondrial enzyme succinic semialdehyde dehydrogenase (SSADH), an enzyme involved in γ-aminobutyric acid metabolism. Succinic semialdehyde dehydrogenase deficiency, resulting from mutations of ALDH5A1, causes a variety of neural and behavioral abnormalities, including strabismus. Variation in ALDH5A1 is likely to contribute to degree of horizontal phoria.

Role of ion channels and subcellular Ca2+ signalling in arachidonic acid induced dilation of pressurised retinal arterioles

Purpose: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Methods: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacological agents inhibiting Ca2+ sparks and oscillations and K+ channels. Subcellular Ca2+ signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Results: AA dilated pressurised retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. AA-induced dilation was associated with an inhibition of subcellular Ca2+ signals. Interventions known to block Ca2+ sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. AA accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous BK currents, but only at positive membrane potentials. Pharmacological inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. AA suppressed L-type Ca2+ currents. Conclusions: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca2+ signalling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca2+ channel activity. AA actions on K+ currents are inconsistent with a model in which K+ channels contribute to the vasodilator effects of AA.

Intravitreal Injection of Normal Saline Induces Retinal Degeneration in the C57BL/6J Mouse

Purpose: To investigate the adverse effect of intravitreal injection of normal saline (NS) and phosphate buffered saline (PBS) in mouse eyes.

Methods: NS or PBS was injected intravitreally into C57BL/6J mouse eyes. Retinal lesions were monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), and histological investigations. Retinal immune gene expression was determined by real-time polymerase chain reaction (PCR). The toxic effect of NS and PBS or retinal protein from NS- or PBS-injected eyes on retinal pigment epithelium (RPE) was tested in B6-RPE-07 mouse RPE cell cultures.

Results: Intravitreal injection of NS dose-dependently induced localized retinal lesion in mice. Histological investigations revealed multiple vacuoles in photoreceptor outer segments and RPE cells. The lesions recovered over time and by 3 weeks post injection the majority of lesions vanished in eyes receiving 1 μl NS. Inflammatory genes, including TNF-α, IL-1β, IL-6, iNOS, and VEGF were upregulated in NS injected eyes. Intravitreal injection of PBS did not cause any pathology. The treatment of B6-RPE07 cells with 30% PBS or 30% NS did not affect RPE viability. However, incubation of 1-μg/ml retinal protein from NS-injected eyes, but not PBS-injected eyes induced RPE cell death.

Conclusion: NS is toxic to the C57BL/6J mouse retina and should not be used as a vehicle for intraocular injection. PBS is not toxic to the retina and is a preferred vehicle.

Translational Relevance: NS is not a physiological solution for intraocular injection in the C57BL/6J mice and questions its suitability for intraocular injection in other species, including human.

Uniformity of visual acuity measures in published studies

PURPOSE. To investigate the methods used in contemporary ophthalmic literature to designate visual acuity (VA). METHODS. Papers in all 2005 editions of five ophthalmic journals were considered. Papers were included if (1) VA, vision, or visual function was mentioned in the abstract and (2) if the study involved age-related macular degeneration, cataract, or refractive surgery. If a paper was selected on the basis of its abstract, the full text of the paper was examined for information on the method of refractive correction during VA testing, type of chart used to measure VA, specifics concerning chart features, testing protocols, and data analysis and means of expressing VA in results. RESULTS. One hundred twenty-eight papers were included. The most common type of charts used were described as logMAR-based. Although most (89.8%) of the studies reported on the method of refractive correction during VA testing, only 58.6% gave the chart design, and less than 12% gave any information whatsoever on chart features or measurement procedures used. CONCLUSIONS. The methods used and the approach to analysis were rarely described in sufficient detail to allow others to replicate the study being reported. Sufficient detail should be given on VA measurement to enable others to duplicate the research. The authors suggest that charts adhering to Bailey-Lovie design principles always be used to measure vision in prospective studies and their use encouraged in clinical settings. The distinction between the terms logMAR, an acuity notation, and Bailey-Lovie or ETDRS as chart types should be adhered to more strictly. Copyright © Association for Research in Vision and Ophthalmology.

FLT1 Genetic Variation Predisposes to Neovascular AMD in Ethnically Diverse Populations and Alters Systemic FLT1 Expression

Purpose: Current understanding of the genetic risk factors for age-related macular degeneration (AMD) is not sufficiently predictive of the clinical course. The VEGF pathway is a key therapeutic target for treatment of neovascular AMD; however, risk attributable to genetic variation within pathway genes is unclear. We sought to identify single nucleotide polymorphisms (SNPs) associated with AMD within the VEGF pathway.
Methods: Using a tagSNP, direct sequencing and meta-analysis approach within four ethnically diverse cohorts, we identified genetic risk present in FLT1, though not within other VEGF pathway genes KDR, VEGFA, or VASH1. We used ChIP and ELISA in functional analysis.
Results: The FLT1 SNPs rs9943922, rs9508034, rs2281827, rs7324510, and rs9513115 were significantly associated with increased risk of neovascular AMD. Each association was more significant after meta-analysis than in any one of the four cohorts. All associations were novel, within noncoding regions of FLT1 that do not tag for coding variants in linkage disequilibrium. Analysis of soluble FLT1 demonstrated higher expression in unaffected individuals homozygous for the FLT1 risk alleles rs9943922 (P = 0.0086) and rs7324510 (P = 0.0057). In silico analysis suggests that these variants change predicted splice sites and RNA secondary structure, and have been identified in other neovascular pathologies. These data were supported further by murine chromatin immunoprecipitation demonstrating that FLT1 is a target of Nr2e3, a nuclear receptor gene implicated in regulating an AMD pathway.
Conclusions: Although exact variant functions are not known, these data demonstrate relevancy across ethnically diverse genetic backgrounds within our study and, therefore, hold potential for global efficacy.

Asymmetric Total Synthesis of (+)-Inthomycin C Via O-Directed Free Radical Alkyne Hydrostannation with Ph3SnH and Catalytic Et3B: Reinstatement of the Zeeck-Taylor (3R)-Structure for (+)-Inthomycin C

A new pathway to (+)-inthomycin C is reported that exploits an O-directed free radical hydrostannation reaction on (−)-12 and a Stille cross-coupling as key steps. Significantly, the latter process was effected on 19 where a gauche-pentane repulsive interaction could interfere. Our stereochemical studies on the alkynol (−)-12 and the enyne (+)-7 confirm that Ryu and Hatakeyama’s (3S)-stereochemical revision of (+)-inthomycin C is invalid and that Zeeck and Taylor’s original (3R)-stereostructure for (+)-inthomycin C is correct.

Experimental Autoimmune Uveoretinitis (EAU)-Related Tissue Damage and Angiogenesis Is Reduced in CCL2−/−CX3CR1gfp/gfp Mice

Purpose: To investigate the roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in experimental autoimmune uveoretinitis (EAU)-mediated retinal tissue damage and angiogenesis.

Methods: The C57BL/6J wild-type (WT) and CCL2−/−CX3CR1gfp/gfp (double knockout [DKO]) mice were immunized with IRBP1-20. Retinal inflammation and tissue damage were evaluated clinically and histologically at different days postimmunization (p.i.). Retinal neovascular membranes were evaluated by confocal microscopy of retinal flat mounts, and immune cell infiltration by flow cytometry.

Results: At day 25 p.i., DKO mice had lower clinical and histological scores and fewer CD45highCD11b+ infiltrating cells compared with WT mice. The F4/80+macrophages constitute 40% and 21% and CD11b+Gr-1+Ly6G+ neutrophils constitute 10% and 22% of retinal infiltrating cells in WT and DKO mice, respectively. At the late stages of EAU (day 60–90 p.i.), DKO and WT mice had similar levels of inflammatory score. However, less structural damage and reduced angiogenesis were detected in DKO mice. Neutrophils were rarely detected in the inflamed retina in both WT and DKO mice. Macrophages and myeloid-derived suppressor cells (MDSCs) accounted for 8% and 3% in DKO EAU retina, and 19% and 10% in WT EAU retina; 71% of infiltrating cells were T/B-lymphocytes in DKO EAU retina and 50% in WT EAU retina.

Conclusions: Experimental autoimmune uveoretinitis–mediated retinal tissue damage and angiogenesis is reduced in CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation is dominated by neutrophils at the acute stage and lymphocytes at the chronic stage in these mice. Our results suggest that CCR2+ and CX3CR1+monocytes are both involved in tissue damage and angiogenesis in EAU.

Retinal Vascular Caliber, Iris Color, and Age-Related Macular Degeneration in the Irish Nun Eye Study

Pupose. To evaluate the relationship between retinal vascular caliber (RVC), iris color and age-related macular degeneration (AMD) in elderly Irish nuns. Methods. Data from 1233 participants in the cross-sectional observational Irish Nun Eye Study were assessed from digital photographs with a standardized protocol using computer-assisted software. Macular images were graded according to the modified Wisconsin age-related maculopathy grading system. Regression models were used to assess associations, adjusting for age, mean arterial blood pressure, body mass index, refraction and fellow RVC. Results. In total, 1122 (91%) participants had gradable retinal images of sufficient quality for vessel assessment (mean age: 76.3 years [range: 56-100 years]). In an unadjusted analysis, we found some support for a previous finding that individuals with blue iris color had narrower retinal venules compared to those with brown iris color (P<0.05) but this was no longer significant after adjustment. AMD status was categorized as no AMD, any AMD and late AMD only. Individuals with any AMD (early or late AMD) had significantly narrower arterioles and venules compared to those with no AMD in an unadjusted analysis but this was no longer significant after adjustment. A non-significant reduced risk of any AMD or late AMD only was observed in association with brown compared to blue iris color, in both unadjusted and adjusted analyses. Conclusions. RVC was not significantly associated with iris color or early/late AMD after adjustment for confounders. A lower but non-significant AMD risk was observed in those with brown compared to blue iris color.

Genetic Background and Light-Dependent Progression of Photoreceptor Cell Degeneration in Prominin-1 Knockout Mice

PURPOSE: Mutations in the Prominin-1 (Prom1) gene are known to cause retinitis pigmentosa and Stargardt disease, both of which are associated with progressive photoreceptor cell death. There are no effective therapies for either disorder. The aim of this study was to investigate the mechanism of the retinal degeneration in Prom1-deficient mouse models.

METHODS: We constructed Prom1 knockout mice with two distinct genetic backgrounds of C57BL/6 and C57BL/6xCBA/NSlc, and investigated the photoreceptor degeneration by means of histology and functional tests.. In addition, we examined the effect of light on the Prom1(-/-) retina by rearing the mice in the normal light/dark cycle and completely dark conditions. Finally, we investigated if the retinoic-acid derivative Fenretinide slowed the pace of retinal degeneration in these mouse models.

RESULTS: The Prom1(-/-)-knockout mice with both backgrounds developed photoreceptor degeneration after eye opening, but the CB57/BL6-background mice developed photoreceptor cell degeneration much faster than the C57BL/6xCBA/NSlc mice, demonstrating genetic background dependency.. Interestingly, our histologic and functional examination showed that the photoreceptor cell degeneration of Prom1-knockout mice was light-dependent, and was almost completely inhibited when the mutant mice were kept in the dark. The Prom1-knockout retina showed strong downregulation of expression of the visual cycle components, Rdh12 and Abca4. Furthermore, administration of Fenretinide, which lowers the level of the toxic lipofuscin, slowed the degeneration of photoreceptor cells.

CONCLUSIONS: These findings improve our understanding of the mechanism of cell death in Prominin-1-related disease and provide evidence that fenretinide may be worth studying in human disease.