Purpose
To analyze mitochondrial DNA (mt DNA) gene mutations in a 19-year-old female patient, who presented with chronic progressive external ophthalmoplegia (CPEO), together with her mother and younger sister.
Methods
The diagnosis of mitochondrial myopathy was made based on clinical and biologic analysis. Histochemical methods were used to detect ragged-red fibers (RRFs) and ragged-blue fibers (RBFs) on a muscle biopsy of the patient. All mitochondrial gene DNA fragments of the patient, her mother, and younger sister were amplified by polymerase chain reaction. The products were sequenced and compared with reference databases.
Results
A novel T1658C mutation and a known A10006G mutation were identified in the mitochondrial tRNAVal gene and the tRNAGly gene, respectively, in the patient, her mother, and younger sister. The T1658C mutation changes the T loop structure of mitochondrial tRNAVal and the A10006G mutation disturbs the D loop of mitochondrial tRNAGly.
Conclusions
The T1658C and A10006G mutations of mtDNA may be responsible for the pathogenesis of the patient with CPEO.
Purpose: To evaluate outcomes of choroidal melanoma patients treated with 125 I or 103 Pd plaque brachytherapy.
Methods and Materials: From 1993 to 2012, our institution treated 160 patients with 103 Pd (56.1%) and 125 patients with 125 I (43.9%) plaque brachytherapy. Tumor outcomes, visual acuity (VA), and toxicity were compared. Multivariate analyses (MVAs) and propensity score analysis were used to help address differences in baseline characteristics.
Results: Median followup was longer for 125 I patients, 52.7 vs. 43.5 months (p < 0.01). At baseline, 103 Pd patients had lower rates of VA worse than 20/200 (4.4% vs. 16%, p = 0.002), T3–T4 tumors (17.5% vs. 32.8%, p = 0.03), and transpupillary thermotherapy use (3.1% vs. 9.6%, p = 0.001). Both 103 Pd and 125 I provided > 90% 3-year overall survival and > 93% 5-year secondary enucleation-free survival. On MVA, radionuclide was not predictive for tumor outcomes. A higher percentage maintained vision better than 20/40 with 103 Pd (63% vs. 35%, p = 0.007) at 3 years. MVA demonstrated 103 Pd radionuclide (odds ratio [OR]: 2.12, p = 0.028) and tumor height ≤5 mm (OR: 2.78, p = 0.017) were associated with VA better than 20/40. Propensity score analysis matched 23 125 I with 107 103 Pd patients. 103 Pd continued to predict better VA at 3 years (OR: 8.10, p = 0.014). On MVA for the development of VA worse than 20/200 or degree of vision loss, radionuclide was not significant. Lower rates of radiation retinopathy were seen with 103 Pd than 125 I (3 years: 47.3% vs. 63.9%, p = 0.016), with radionuclide significant in MVA.
Conclusions: Both 125 I and 103 Pd achieve excellent tumor control. An increased probability of long-term VA better than 20/40 and reduced risk of radiation retinopathy is associated with 103 Pd.
Background: Cumulative oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays key roles in retinal antioxidant and detoxification responses. The purposes of this study were to determine whether NRF2-deficient mice would develop AMD-like retinal pathology with aging and to explore the underlying mechanisms.
Methods and Findings: Eyes of both wild type and Nrf2-/- mice were examined in vivo by fundus photography and electroretinography (ERG). Structural changes of the outer retina in aged animals were examined by light and electron microscopy, and immunofluorescence labeling. Our results showed that Nrf2-/- mice developed age-dependent degenerative pathology in the retinal pigment epithelium (RPE). Drusen-like deposits, accumulation of lipofuscin, spontaneous choroidal neovascularization (CNV) and sub-RPE deposition of inflammatory proteins were present in Nrf2-/- mice after 12 months. Accumulation of autophagy-related vacuoles and multivesicular bodies was identified by electron microcopy both within the RPE and in Bruch's membrane of aged Nrf2-/- mice.
Conclusions: Our data suggest that disruption of Nfe2l2 gene increased the vulnerability of outer retina to age-related degeneration. NRF2-deficient mice developed ocular pathology similar to cardinal features of human AMD and deregulated autophagy is likely a mechanistic link between oxidative injury and inflammation. The Nrf2-/- mice can provide a novel model for mechanistic and translational research on AMD.
Purpose: To investigate the effect of bevacizumab (Avastin; Genentech, San Francisco, CA, USA) on vascular endothelial growth factor (VEGF) expression and inflammation in fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR).
Materials and Methods: Fibrovascular membranes from 19 eyes of 18 patients with PDR were studied using immunohistochemistry and analyzed in the following 3 groups; group 1: 4 inactive PDR eyes, group 2: 10 active PDR eyes treated preoperatively with adjunctive intravitreal bevacizumab, group 3: five active PDR eyes not treated preoperatively with bevacizumab. Immunohistochemical staining for VEGF, CD31 and CD68 were done.
Results: The immunoreactivity to VEGF and CD 31-positive blood vessels was significantly higher in membranes from group 3 than group 1 (p = 0.007 for VEGF, 0.013 for CD 31-positive vessels). Intravitreal bevacizumab caused a reduction in VEGF expression and vascular densities in 4 out of 10 (40%) excised membranes from eyes with PDR. However, six membranes (60%) in group 2 still demonstrated relatively strong VEGF expression and high vascular density. Infiltration of macrophages was observed in 16 out of the 19 membranes, and the density of macrophages was increased in group 2 compared with group 1 (p = 0.043).
Conclusion: Intravitreal bevacizumab injections caused some reduction in VEGF expression and vascular densities in a limited number of active PDR patients. A single intravitreal bevacizumab injection may not be enough to induce complete blockage of VEGF and pathologic neovascularization in active PDR patients. Repeated injections, panretinal photocoagulation and/or PPV may be necessary following intravitreal bevacizumab to reinforce the anti-VEGF effect of the drug.
Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression.
In the mammalian retina, dopamine binding to the dopamine D4 receptor affects a light-sensitive pool of cyclic AMP by negatively coupling to the type 1 adenylyl cyclase. The type 1 adenylyl cyclase is the primary enzyme controlling cyclic AMP production in dark-adapted photoreceptors. A previous study demonstrated that expression of the gene encoding the type 1 adenylyl cyclase, Adcy1, is down regulated in mice lacking Drd4, the gene encoding the dopamine D4 receptor. The present investigation provides evidence that dopamine D4 receptor activation entrains the circadian rhythm of Adcy1 mRNA expression. Diurnal and circadian rhythms of Drd4 and Adcy1 mRNA levels were observed in wild type mouse retina. Also, rhythms in the Ca2+-stimulated adenylyl cyclase activity and cyclic AMP levels were observed. However, these rhythmic activities were damped or undetectable in mice lacking the dopamine D4 receptor. Pharmacologically activating the dopamine D4 receptor 4 hrs before its normal stimulation at light onset in the morning advances the phase of the Adcy1 mRNA expression pattern. These data demonstrate that stimulating the dopamine D4 receptor is essential in maintaining the normal rhythmic production of the type 1 adenylyl cyclase from transcript to enzyme activity. Thus, dopamine/dopamine D4 receptor signaling is a novel zeitgeber that entrains the rhythm of Adcy1 expression and, consequently, modulates the rhythmic synthesis of cyclic AMP in mouse retina.
Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca2+ concentrations ([Ca2+]i) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5–7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K+-stimulated uptake of 45Ca2+ and [Ca2+]i, measured with the Ca2+-sensitive fluorescent dye fura-2 AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell permeable analog of cAMP, had no effect on inhibition of K+-stimulated 45Ca2+ influx or [Ca2+]i by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K+, which requires Ca2+ influx through voltage-gated Ca2+ channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca2+/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca2+]i.
by
Qing Zhang;
Micah A. Chrenek;
Shagun Bhatia;
Alia Rashid;
Salma Ferdous;
Kevin J. Donaldson;
Henry Skelton;
Wenfei Wu;
Thonnie Rose O. See;
Yi Jiang;
Nupur Dalal;
John Nickerson;
Hans Grossniklaus
Purpose: To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD. Methods: Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images. Results: Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0-51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60-93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (<63 µm), medium (63.0-124 µm), and large ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>≥</mml:mo></mml:math> 125 µm) drusen. The RPE was arranged in rosette-like structures overlying small drusen, attenuated overlying medium-sized drusen, and consisted of large multinucleated cells overlying large drusen. The RPE in the area of geographic atrophy was attenuated and depigmented. Conclusions: Confocal images of flatmounts from eyes with AMD showed RPE patterns overlying various types of drusen and geographic atrophy that correlated with histologic characteristics. We propose RPE repair mechanisms that may result in the patterns that we observed.
Despite the high prevalence and public health impact of refractive errors, the mechanisms responsible for ametropias are poorly understood. Much evidence now supports the concept that the retina is central to the mechanism(s) regulating emmetropization and underlying refractive errors. Using a variety of pharmacologic methods and well-defined experimental eye growth models in laboratory animals, many retinal neurotransmitters and neuromodulators have been implicated in this process. Nonetheless, an accepted framework for understanding the molecular and/or cellular pathways that govern postnatal eye development is lacking. Here, we review two extensively studied signaling pathways whose general roles in refractive development are supported by both experimental and clinical data: acetylcholine signaling through muscarinic and/or nicotinic acetylcholine receptors and retinal dopamine pharmacology. The muscarinic acetylcholine receptor antagonist atropine was first studied as an anti-myopia drug some two centuries ago, and much subsequent work has continued to connect muscarinic receptors to eye growth regulation. Recent research implicates a potential role of nicotinic acetylcholine receptors; and the refractive effects in population surveys of passive exposure to cigarette smoke, of which nicotine is a constituent, support clinical relevance. Reviewed here, many puzzling results inhibit formulating a mechanistic framework that explains acetylcholine's role in refractive development. How cholinergic receptor mechanisms might be used to develop acceptable approaches to normalize refractive development remains a challenge. Retinal dopamine signaling not only has a putative role in refractive development, its upregulation by light comprises an important component of the retinal clock network and contributes to the regulation of retinal circadian physiology. During postnatal development, the ocular dimensions undergo circadian and/or diurnal fluctuations in magnitude; these rhythms shift in eyes developing experimental ametropia. Long-standing clinical ideas about myopia in particular have postulated a role for ambient lighting, although molecular or cellular mechanisms for these speculations have remained obscure. Experimental myopia induced by the wearing of a concave spectacle lens alters the retinal expression of a significant proportion of intrinsic circadian clock genes, as well as genes encoding a melatonin receptor and the photopigment melanopsin. Together this evidence suggests a hypothesis that the retinal clock and intrinsic retinal circadian rhythms may be fundamental to the mechanism(s) regulating refractive development, and that disruptions in circadian signals may produce refractive errors. Here we review the potential role of biological rhythms in refractive development. While much future research is needed, this hypothesis could unify many of the disparate clinical and laboratory observations addressing the pathogenesis of refractive errors.
In recent years, there has been a growing interest for using mouse models in refractive development and myopia research. The crystalline lens is a critical optical component of the mouse eye that occupies greater than 50% of the ocular space, and significant increases in thickness with age. However, changes in refractive index of the mouse crystalline lens are less known. In this study, we examined the changes in thickness and refractive index of the mouse crystalline lens for two different strains, wild-type (WT) and a nyx mutant (nob) over the course of normal visual development or after form deprivation. Refractive index and lens thickness measurements were made on exvivo lenses using spectral domain optical coherence tomography (SD-OCT). Comparison of refractive index measurements on 5 standard ball lenses using the SD-OCT and their known refractive indices (manufacturer provided) indicated good precision (intra-class correlation coefficient, 0.998 and Bland-Altman coefficient of repeatability, 0.116) of the SD-OCT to calculate mouse lens refractive index exvivo. During normal visual development, lens thickness increased significantly with age for three different cohorts of mice, aged 4 (average thickness from both eyes; WT: 1.78±0.03, nob: 1.79±0.08mm), 10 (WT: 2.02±0.05, nob: 2.01±0.04mm) and 16 weeks (WT: 2.12±0.06, nob: 2.09±0.06mm, p<0.001). Lens thickness was not significantly different between the two strains at any age (p=0.557). For mice with normal vision, refractive index for isolated crystalline lenses in nob mice was significantly greater than WT mice (mean for all ages; WT: 1.42±0.01, nob: 1.44±0.001, p<0.001). After 4 weeks of form deprivation to the right eye using a skull-mounted goggling apparatus, a thinning of the crystalline lens was observed in both right and left eyes of goggled animals compared to their naïve controls (average from both the right and the left eye) for both strains (p=0.052). In form deprived mice, lens refractive index was significantly different between the goggled animals and non-goggled naïve controls in nob mice, but not in WT mice (p=0.009). Both eyes of goggled nob mice had significantly greater lens refractive index (goggled, 1.49±0.01; opposite, 1.47±0.03) compared to their naïve controls (1.45±0.02, p<0.05). The results presented here suggest that there are genetic differences in the crystalline lens refractive index of the mouse eye, and that the lens refractive index in mice significantly increase with form deprivation. Research applications requiring precise optical measurements of the mouse eye should take these lens refractive indices into account when interpreting SD-OCT data.