Purpose.

Tissue glue containing fibrinogen (FIB) and riboflavin (RF), upon exposure to long wavelength ultraviolet light (UVA, 365 nM) has been proposed potentially to solve long-standing problems presented by corneal wound and epithelial ingrowth side-effects from laser-assisted in situ keratomileuis (LASIK). Data presented in a previous study demonstrated an ability of FIB + RF + UVA to adhere two stromal surfaces; however, to our knowledge no molecular mechanisms have been proposed to account for interactions occurring between corneal extracellular matrix (ECM) and tissue glue molecules. Here, we document several covalent and noncovalent interactions between these classes of macromolecules.

Methods.

SDS-PAGE and Western blot techniques were used to identify covalent interactions between tissue glue molecules and corneal ECM molecules in either the presence or absence of RF and UVA, in vitro and ex vivo. Surface plasmon resonance (SPR) was used to characterize noncovalent interactions, and obtain ka, kd, and KD binding affinity values.

Results.

SDS-PAGE and Western blot analyses indicated that covalent interactions occurred between neighboring FIB molecules, as well as between FIB and collagen type I (Coll-I) proteins (in vitro and ex vivo). These interactions occurred only in the presence of RF and UVA. SPR data demonstrated the ability of FIB to bind noncovalently to corneal stroma molecules, Coll-I, decorin, dermatan sulfate, and corneal basement membrane molecules, laminin and heparan sulfate – only in the presence of Zn2+.

Conclusions.

Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA.

FIB + RF + UVA has been shown to act as a tissue glue in the corneal stroma. This article elucidates the covalent and noncovalent molecular mechanisms of the FIB + RF + UVA treatment.

Purpose.

Visibility of low–spatial frequency stimuli improves when their contrast is modulated at 5 to 10 Hz compared with stationary stimuli. Therefore, temporal modulations of visual objects could enhance the performance of low vision patients who primarily perceive images of low–spatial frequency content. We investigated the effect of retinal-image jitter on word recognition speed and facial emotion recognition in subjects with central visual impairment.

Methods.

Word recognition speed and accuracy of facial emotion discrimination were measured in volunteers with AMD under stationary and jittering conditions. Computer-driven and optoelectronic approaches were used to induce retinal-image jitter with duration of 100 or 166 ms and amplitude within the range of 0.5 to 2.6° visual angle. Word recognition speed was also measured for participants with simulated (Bangerter filters) visual impairment.

Results.

Text jittering markedly enhanced word recognition speed for people with severe visual loss (101 ± 25%), while for those with moderate visual impairment, this effect was weaker (19 ± 9%). The ability of low vision patients to discriminate the facial emotions of jittering images improved by a factor of 2. A prototype of optoelectronic jitter goggles produced similar improvement in facial emotion discrimination. Word recognition speed in participants with simulated visual impairment was enhanced for interjitter intervals over 100 ms and reduced for shorter intervals.

Conclusions.

Results suggest that retinal-image jitter with optimal frequency and amplitude is an effective strategy for enhancing visual information processing in the absence of spatial detail. These findings will enable the development of novel tools to improve the quality of life of low vision patients.

The ability of low vision patients to read text and recognize facial emotions improves when the retinal image is jittered. Image jitter has the potential to supplement other conventional techniques used in low vision rehabilitation.

Purpose.

To investigate, using multifocal electroretinography (mfERG) and optical coherence tomography (OCT), potential spatial associations between local neuroretinal function and local retinal thickness in patients with diabetes.

Methods.

Forty-five patients without retinopathy (10 with Type 1 diabetes; 35 with Type 2 diabetes; 49.9 ± 10.9 years old) and 29 age-similar controls (47.0 ± 12.8 years old) were studied. N1-P1 amplitude (AMP) and P1 implicit time (IT) of mfERGs within the central approximately 20° diameter were compared to spatially corresponding full retinal thickness measurements acquired by Stratus OCT3. AMP and IT were converted to Z-scores and retinal thickness was converted to percentile values. Local abnormalities were defined as P ≤ 0.023. Subject group differences were examined using t-tests. Retinal thickness was compared to mfERGs to determine spatial associations.

Results.

Average retinal thicknesses were similar for all subject groups. The Type 1 group and controls had similar IT and AMP. The Type 2 group had reduced AMP and longer IT than the controls and the Type 1 group (P < 0.001). Local associations between retinal thickness and mfERGs were not significant within any subject group or individuals, even for abnormal locations (P ≥ 0.09). Abnormalities in most measures were greater in the patient groups than in the controls (P < 0.008) except retinal thinning in the Type 1 group.

Conclusions.

Local neuroretinal function is not associated with full retinal thickness measured locally in patients with diabetes and no retinopathy, even in abnormal locations. Full retinal thickness measured locally by OCT is not a surrogate for mfERGs in early diabetes. Neuroretinal function in Type 2 diabetes is worse than in Type 1 diabetes and controls. Fewer subjects in the Type 1 group could be a potential limitation.

We examine the relationship between neuroretinal function tested using multifocal electroretinogram and full retinal thickness assessed locally using optical coherence tomography. Retinal function is not locally associated with full retinal thickness in patients with diabetes and no retinopathy, even in abnormal locations.

Purpose.

Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears.

Methods.

Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed.

Results.

Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal–reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA.

Conclusions.

Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

Protein evidence for the first nonprimate lacritin is described.

Purpose.

We determined whether the street-crossing decisions of subjects with age-related macular degeneration (AMD) were as accurate and precise as those made by young and older subjects with normal vision.

Methods.

Street-crossing decisions in 13 AMD subjects, and 20 young and 20 older control subjects with normal vision were measured along an un-signalized street for nine different gap times. After calculating the discriminability (d') of the street-crossing decision variable for all gap pairs and entering these d' values into a one-dimensional scaling model, the means of each distribution of the decision variable relative to a “center of gravity” were estimated and plotted against gap time. The resultant plot was a nonlinear function. Street-crossing decision accuracy was computed for each subject as the difference between the x-intercept of the nonlinear function (tCOG) and subjects' measured street-crossing time. Street-crossing decision-making precision was computed as the value of the slope of the nonlinear function at tCOG.

Results.

We found that all subjects were precise in their street-crossing decisions (P = 0.55). Significant differences in street-crossing accuracy were found as a function of age (P = 0.003). Compared to either the older normally-sighted (P = 0.018) or AMD (P = 0.019) subjects, the young normally-sighted subjects made the least accurate street-crossing decisions. No significant difference in accuracy was found between the AMD and age-matched normally-sighted subjects (P = 0.90).

Conclusions.

Our data suggested that age and mild central vision loss did not affect significantly a subject's precision in their street-crossing decisions. Age, but not mild central vision loss, significantly affected a subject's accuracy in their street-crossing decisions.

We found that AMD subjects were just as accurate and precise as age-matched normally-sighted subjects in making street-crossing decisions. Our results also indicated that age had a significant effect on street-crossing decision-making accuracy but not on precision.

Purpose.

To test the intra- and intersubject reproducibility of brain activation patterns that underlie visually guided saccades and word recognition in normally sighted subjects and patients with macular degeneration using functional magnetic resonance imaging (fMRI).

Methods.

Ten normally sighted subjects and five patients with macular degeneration were asked to perform two visually guided saccade tasks and two word-recognition tasks during fMRI with behavioral monitoring. The fMRI measurements were repeated three times at intervals of at least 4 weeks between sessions. The intrasubject reproducibility of the brain activation patterns was examined in a model-independent manner by comparing the distributions of activation across the frontal, parietal, temporal, and occipital brain lobes using Intraclass Correlation Coefficients (ICCs). Intersubject reproducibility was examined by repeated-measure ANOVA.

Results.

Control subjects showed overall higher intrasubject reproducibility of brain activation patterns (75% ICCs > 0.5) than that of patients with macular degeneration (56% ICCs > 0.5). The intrasubject reproducibility for the patients improved when the target location was fixed, as in the word-recognition tasks (75% ICCs > 0.5), compared with the visually saccade tasks (37% ICCs > 0.5). Intersubject variability of brain activation patterns was strikingly high for both the control and patient groups.

Conclusions.

The fMRI method can serve as a reliable within-subjects measure of brain activation that has potential for measuring longitudinal changes in brain networks associated with rehabilitation training. Striking intersubject variability reflected at the level of lobes of the brain among control subjects with similar behavioral performance, suggests individual analysis is necessary when implementing longitudinal brain activation studies.

The fMRI method can serve as a reliable within-subjects measure of brain activation that has potential for measuring longitudinal changes in brain networks associated with rehabilitation training.

Purpose.

Age-Related Eye Disease Study 2 (AREDS2) is a randomized, placebo-controlled study designed to determine whether supplementation with 10 mg of lutein and 2 mg of zeaxanthin per day can slow the rate of progression of age-related macular degeneration (AMD). Although some biomarkers of response to carotenoid supplementation such as serum concentrations are part of the AREDS2 protocol, measurement of carotenoid concentrations in the eye and other tissues is not. In this approved ancillary study, macular pigment optical density (MPOD), macular pigment distributions, and skin carotenoid levels at enrollment and at each annual visit were measured to assess baseline carotenoid status and to monitor response to assigned interventions.

Methods.

All subjects enrolled at the Moran Eye Center had MPOD and macular pigment spatial distributions measured by dual-wavelength autofluorescence imaging and total skin carotenoids measured by resonance Raman spectroscopy.

Results.

Baseline MPOD in enrolled subjects was unusually high relative to an age-matched control group that did not consume carotenoid supplements regularly, consistent with the high rate of habitual lutein and zeaxanthin consumption in Utah AREDS2 subjects prior to enrollment. MPOD did not correlate with serum or skin carotenoid measurements.

Conclusions.

Useful information is provided through this ancillary study on the ocular carotenoid status of AREDS2 participants in the target tissue of lutein and zeaxanthin supplementation: The macula. When treatment assignments are unmasked at the conclusion of the study, unique tissue-based insights will be provided on the progression of AMD in response to long-term, high-dose carotenoid supplementation versus diet alone. (ClinicalTrials.gov number, NCT00345176.)

Baseline macular pigment measurements at the Utah AREDS2 site were unusually high relative to an age-matched control group that did not consume carotenoid supplements regularly, consistent with the high rate of habitual lutein and zeaxanthin consumption in Utah AREDS2 subjects prior to enrollment.

Purpose.

To assess structural, functional, and visual behavioral relationships in mutant rhodopsin transgenic (Tg) rats and to determine whether early optokinetic tracking (OKT) visual experience, known to permanently elevate visual thresholds in normal rats, can enhance vision in rats with photoreceptor degeneration.

Methods.

Eight lines of pigmented Tg rats and RCS rats were used in this study. OKT thresholds were tested at single ages (1, 2, 3, 4, and 6 months) in naïve groups of rats, or daily in groups that began at eye-opening (P15) or 10 days later (P25). Electroretinogram (ERG) response amplitudes were recorded after OKT testing, and outer nuclear layer (ONL) thickness measurements were then obtained.

Results.

OKT thresholds, when measured at a single time point in naïve Tg lines beginning at P30, did not decline until months after significant photoreceptor loss. Daily testing of Tg lines resulted mostly with OKT thresholds inversely related to photoreceptor degeneration, with rapid degenerations resulting in sustained OKT thresholds for long periods despite the rapid photoreceptor loss. Slower degenerations resulted in rapid decline of thresholds, long before the loss of most photoreceptors, which was even more pronounced when daily testing began at eye opening. This amplified loss of function was not a result of testing-induced damage to the rod or cone photoreceptors, as ERG amplitudes and ONL thicknesses were the same as untested controls.

Conclusions.

The unexpected lack of correlation of OKT testing with photoreceptor degeneration in the Tg rats emphasizes the need in behavioral therapeutic studies for careful analysis of visual thresholds of experimental animals prior to therapeutic intervention.

Optomotor testing accelerated visual decline in rhodopsin mutant rats, but not in RCS rats. The degree of vision loss did not correlate with electrophysiological or histological indices. Therefore, care should be taken when examining the visual performance of rodent models of retinal degeneration.

Purpose.

Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).

Methods.

We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of αvβ5 integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.

Results.

Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.

Conclusions.

Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients.

In this manuscript, we describe the development and application of a novel, rapid, and quantitative flow cytometry-based assay to assess photoreceptor outer segment phagocytosis by retinal pigment epithelium (RPE) cells in vitro and have used it to evaluate and compare human induced pluripotent stem (iPS) cell-derived RPE cells with ARPE-19 and human fetal RPE.

Purpose.

Neuron-specific enolase (NSE) is a biomarker for neuronal stress. Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease affecting retinal ganglion cells (RGC). These RGCs and their axons in the retinal nerve fiber layer (RNFL) and optic nerve head may show subclinical pathology in unaffected mutation carriers, or undergo cell death in affected patients. We hypothesize that increased levels of blood NSE may characterize LHON carriers as a biomarker of ongoing RGC stress.

Methods.

Serum was obtained from 74 members of a Brazilian pedigree with LHON carrying the homoplasmic 11778/ND4 mitochondrial DNA mutation. Classified by symptoms and psychophysical metrics, 46/74 patients were unaffected mutation “carriers,” 14/74 were “affected,” and 14/74 were “off-pedigree” controls. Serum NSE levels were determined by ELISA specific for the γ subunit of NSE.

Results.

Serum NSE concentrations in carriers (27.17 ± 39.82 μg/L) were significantly higher than affected (5.66 ± 4.19 μg/L; P = 0.050) and off-pedigree controls (6.20 ± 2.35 μg/L; P = 0.047). Of the 14/46 (30.4 %) carriers with significantly elevated NSE levels (mean = 75.8 ± 42.3 μg/L), 9/14 (64.3%) were male. Furthermore, NSE levels were nearly three times greater in asymptomatic male carriers (40.65 ± 51.21 μg/L) than in asymptomatic female carriers (15.85 ± 22.27 μg/L; P = 0.034).

Conclusions.

Serum NSE levels are higher in LHON carriers compared with affected and off-pedigree individuals. A subgroup of mostly male carriers had significantly elevated serum NSE levels. Thus, male carriers are at higher risk for LHON-related neuronal stress.

Asymptomatic carriers of LHON have higher serum NSE levels than affected and off-pedigree counterparts. Increased levels of serum NSE may be used as a biomarker to characterize RGC stress in LHON carriers.

Purpose.

We examined the association between caffeine and caffeinated beverage consumption in relation to the risk of exfoliation glaucoma or exfoliation glaucoma suspect (EG/EGS).

Methods.

We followed 78,977 women from the Nurses' Health Study (NHS) and 41,202 men from the Health Professionals Follow-up Study (HPFS) who were at least 40 years of age, did not have glaucoma, and reported undergoing eye examinations from 1980 (NHS) or 1986 (HPFS) to 2008. Information on consumption of caffeine-containing beverages and potential confounders were repeatedly ascertained in validated follow-up questionnaires. Confirmation with medical record review revealed 360 incident EG/EGS cases. Multivariate rate ratios (RRs) for EG/EGS were calculated in each cohort and then pooled using meta-analytic techniques.

Results.

Compared with participants whose cumulatively updated total caffeine consumption was <125 mg/day, participants who consumed ≥500 mg/day had a trend toward increased risk of EG/EGS that was not statistically significant (RR = 1.43; 95% confidence interval [CI], 0.98–2.08); P trend = 0.06). Compared to abstainers, those who drank ≥3 cups of caffeinated coffee daily were at increased risk of EG/EGS (RR = 1.66; 95% CI, 1.09–2.54; P trend = 0.02). These results were not materially altered after adjustment for total fluid intake. Associations were stronger among women with a family history of glaucoma (P interaction = 0.06 for coffee; P interaction = 0.03 for caffeine). We did not find associations with consumption of other caffeinated products (caffeinated soda, caffeinated tea, decaffeinated coffee or chocolate) and risk of EG/EGS (P trend ≥0.31).

Conclusions.

We observed a positive association between heavier coffee consumption with risk of EG/EGS in this large prospective study.

In a large 28-year-old prospective study including 360 incident cases, caffeinated coffee consumption was significantly associated with an increased risk of exfoliation glaucoma or exfoliation glaucoma suspect. The association between caffeine consumption was particularly strong among those with a positive family history of glaucoma

Purpose.

We evaluated the effect of imposing negative and positive defocus simultaneously on the eye growth and refractive state of the common marmoset, a New World primate that compensates for either negative and positive defocus when they are imposed individually.

Methods.

Ten marmosets were reared with multizone contact lenses of alternating powers (−5 diopters [D]/+5 D), 50:50 ratio for average pupil of 2.80 mm over the right eye (experimental) and plano over the fellow eye (control) from 10 to 12 weeks. The effects on refraction (mean spherical equivalent [MSE]) and vitreous chamber depth (VC) were measured and compared to untreated, and −5 D and +5 D single vision contact lens-reared marmosets.

Results.

Over the course of the treatment, pupil diameters ranged from 2.26 to 2.76 mm, leading to 1.5 times greater exposure to negative than positive power zones. Despite this, at different intervals during treatment, treated eyes were on average relatively more hyperopic and smaller than controls (experimental-control [exp-con] mean MSE ± SE +1.44 ± 0.45 D, mean VC ± SE −0.05 ± 0.02 mm) and the effects were similar to those in marmosets raised on +5 D single vision contact lenses (exp-con mean MSE ± SE +1.62 ± 0.44 D. mean VC ± SE −0.06 ± 0.03 mm). Six weeks into treatment, the interocular growth rates in multizone animals were already lower than in −5 D-treated animals (multizone −1.0 ± 0.1 μm/day, −5 D +2.1 ± 0.9 μm/day) and did not change significantly throughout treatment.

Conclusions.

Imposing hyperopic and myopic defocus simultaneously using concentric contact lenses resulted in relatively smaller and less myopic eyes, despite treated eyes being exposed to a greater percentage of negative defocus. Exposing the retina to combined dioptric powers with multifocal lenses that include positive defocus might be an effective treatment to control myopia development or progression.

Purpose.

To assess the prevalence of peripheral fundus autofluorescence (FAF) abnormalities in a variety of diseases seen at a tertiary retina clinic.

Methods.

We conducted a retrospective review of cases seen at the Doheny Eye Institute between November 2009 and May 2011, who had ultra-widefield FAF and pseudocolor imaging performed on new models of scanning laser ophthalmoscopes. Patients with a history of previous therapies that could alter the FAF findings, including vitrectomy, cryotherapy, laser photocoagulation, or photodynamic therapy, were excluded from the analysis. Based on their primary diagnosis the eyes were grouped into nine disease categories: age-related macular degeneration, central serous retinopathy, dystrophy, inflammatory disorders, ocular tumor, retinal vascular disorders, other, normal, and unknown. All FAF and accompanying pseudocolor images were reviewed independently by two reading center–certified graders.

Results.

A total of 470 eyes of 248 patients were included for analysis of which 461 eyes had images of sufficient quality for grading. The prevalence of peripheral findings was 65.5% (n = 302) for FAF images and 68.5% (n = 316) for the pseudocolor images (P < 0.001). The prevalence of peripheral abnormalities differed significantly between the disease categories ranging from 18.5% to 82.2% for FAF and 18.5% to 82.4% for pseudocolor images.

Conclusions.

Peripheral FAF abnormalities are frequent and readily revealed by FAF imaging. Interestingly, even cases with presumably macular disease demonstrated a high prevalence of peripheral findings. Further investigation in prospective studies is warranted.

Peripheral abnormalities on ultra-widefield autofluorescence images are common in a large variety of diseases and reliably identifiable. There is a high correspondence between changes seen on autofluorescence and pseudocolor images. Peripheral changes in ultra-widefield imaging are a common finding in a variety of different diseases.

Purpose.

We evaluated Progression of Patterns (POP) for its ability to identify progression of glaucomatous visual field (VF) defects.

Methods.

POP uses variational Bayesian independent component mixture model (VIM), a machine learning classifier (MLC) developed previously. VIM separated Swedish Interactive Thresholding Algorithm (SITA) VFs from a set of 2,085 normal and glaucomatous eyes into nine axes (VF patterns): seven glaucomatous. Stable glaucoma was simulated in a second set of 55 patient eyes with five VFs each, collected within four weeks. A third set of 628 eyes with 4,186 VFs (mean ± SD of 6.7 ± 1.7 VFs over 4.0 ± 1.4 years) was tested for progression. Tested eyes were placed into suspect and glaucoma categories at baseline, based on VFs and disk stereoscopic photographs; a subset of eyes had stereophotographic evidence of progressive glaucomatous optic neuropathy (PGON). Each sequence of fields was projected along seven VIM glaucoma axes. Linear regression (LR) slopes generated from projections onto each axis yielded a degree of confidence (DOC) that there was progression. At 95% specificity, progression cutoffs were established for POP, visual field index (VFI), and mean deviation (MD). Guided progression analysis (GPA) was also compared.

Results.

POP identified a statistically similar number of eyes (P > 0.05) as progressing compared with VFI, MD, and GPA in suspects (3.8%, 2.7%, 5.6%, and 2.9%, respectively), and more eyes than GPA (P = 0.01) in glaucoma (16.0%, 15.3%, 12.0%, and 7.3%, respectively), and more eyes than GPA (P = 0.05) in PGON eyes (26.3%, 23.7%, 27.6%, and 14.5%, respectively).

Conclusions.

POP, with its display of DOC of progression and its identification of progressing VF defect pattern, adds to the information available to the clinician for detecting VF progression.

Progression of Patterns (POP) is a novel machine learning classifier (MLC) algorithm, based on our modification of independent component analysis (ICA), for determining if an eye is stable or shows progression of glaucomatous visual field (VF) defects. This mathematical approach seeks to avoid human bias.

Purpose.

To develop an automated method for the detection of retinal hemorrhages on color fundus images to characterize malarial retinopathy, which may help in the assessment of patients with cerebral malaria.

Methods.

A fundus image dataset from 14 patients (200 fundus images, with an average of 14 images per patient) previously diagnosed with malarial retinopathy was examined. We developed a pattern recognition–based algorithm, which extracted features from image watershed regions called splats (tobogganing). A reference standard was obtained by manual segmentation of hemorrhages, which assigned a label to each splat. The splat features with the associated splat label were used to train a linear k-nearest neighbor classifier that learnt the color properties of hemorrhages and identified the splats belonging to hemorrhages in a test dataset. In a crossover design experiment, data from 12 patients were used for training and data from two patients were used for testing, with 14 different permutations; and the derived sensitivity and specificity values were averaged.

Results.

The experiment resulted in hemorrhage detection sensitivities in terms of splats as 80.83%, and in terms of lesions as 84.84%. The splat-based specificity was 96.67%, whereas for the lesion-based analysis, an average of three false positives was obtained per image. The area under the receiver operating characteristic curve was reported as 0.9148 for splat-based, and as 0.9030 for lesion-based analysis.

Conclusions.

The method provides an automated means of detecting retinal hemorrhages associated with malarial retinopathy. The results matched well with the reference standard. With further development, this technique may provide automated assistance for screening and quantification of malarial retinopathy.

A supervised hemorrhage recognition method is proposed for automated detection of retinal hemorrhages to characterize malarial retinopathy, which may help in the assessment of patients with cerebral malaria. The results of a crossover design experiment match well with the reference standard.

Purpose.

The purpose of this study was to evaluate the anti-inflammatory effect of ethyl pyruvate (EP) in a mouse model of lipopolysaccharide (LPS)-induced corneal inflammation.

Methods.

LPS was injected intrastromally into the corneas of C57BL/6 mice followed by treatment with a solution of 2.5% EP in 0.2% hydroxypropyl methylcellulose (HPMC) every 90 minutes during the course of 12 hours. Prednisolone acetate 1% solution (PRED FORTE) was used as a positive control. Mice were sacrificed after 3 days, and corneas were examined by in vivo confocal microscopy and analyzed for infiltrated cells by flow cytometry. Gr-1, TNF-α, and pNF-κB-p65 were detected immunohistochemically, and TNF-α, IL-6, and IL-1β levels were quantified by ELISA.

Results.

LPS-induced haze in mice corneas was decreased by 2-fold upon EP treatment; however, it was not changed upon PRED FORTE treatment. Flow cytometry and immunohistochemistry showed infiltration of leukocytes in the LPS-treated corneas; among the infiltrated cells, neutrophils (Gr-1+ and CD11b+) and macrophages (F4/80+ and CD11b+) were 3403.4- and 4.5-fold higher in number, respectively, than in vehicle-treated control corneas. EP or PRED FORTE treatment of LPS-injected corneas decreased the number of neutrophils 7.5- and 7.2-fold and macrophages by 5.6- and 3.5-fold, respectively. Both EP and PRED FORTE decreased TNF-α and IL-6 expression considerably, and to a lesser extent IL-1β expression, in the LPS-treated corneas.

Conclusions.

The present study demonstrated that EP reduces LPS-induced inflammation in the cornea and thus may have a potential therapeutic application in the inhibition of corneal inflammation.

The anti-inflammatory effect of ethyl pyruvate (EP) in a mouse model of lipopolysaccharide (LPS)-induced corneal inflammation was evaluated. EP reduced LPS-induced infiltration of inflammatory cells and cytokine levels; thus it has a potential therapeutic use in the inhibition of corneal inflammation. The present study demonstrated that EP reduces LPS-induced inflammation in the cornea in mice as effectively as prednisolone (PRED FORTE). Therefore, EP may have a potential therapeutic application as a nonsteroidal anti-inflammatory chemotherapeutic agent.

Purpose.

AMD results in loss of central vision and a dependence on low-resolution peripheral vision. While many image enhancement techniques have been proposed, there is a lack of quantitative comparison of the effectiveness of enhancement. We developed a natural visual search task that uses patients' eye movements as a quantitative and functional measure of the efficacy of image modification.

Methods.

Eye movements of 17 patients (mean age = 77 years) with AMD were recorded while they searched for target objects in natural images. Eight different image modification methods were implemented and included manipulations of local image or edge contrast, color, and crowding. In a subsequent task, patients ranked their preference of the image modifications.

Results.

Within individual participants, there was no significant difference in search duration or accuracy across eight different image manipulations. When data were collapsed across all image modifications, a multivariate model identified six significant predictors for normalized search duration including scotoma size and acuity, as well as interactions among scotoma size, age, acuity, and contrast (P < 0.05). Additionally, an analysis of image statistics showed no correlation with search performance across all image modifications. Rank ordering of enhancement methods based on participants' preference revealed a trend that participants preferred the least modified images (P < 0.05).

Conclusions.

There was no quantitative effect of image modification on search performance. A better understanding of low- and high-level components of visual search in natural scenes is necessary to improve future attempts at image enhancement for low vision patients. Different search tasks may require alternative image modifications to improve patient functioning and performance.

We examined the effect of image modification on visual search performance in patients with central vision loss from AMD. We found no significant effect on performance measures across eight different image modifications.

Purpose.

Several small proteomic studies suggest that the prosecretory tear protein lacritin may be selectively downregulated in dry eye syndrome and in blepharitis, yet little information is available about normal baseline levels. This study assessed lacritin levels in tears from healthy individuals and addressed whether they differ according to sex, age, or time of day.

Methods.

Rabbit antibodies against lacritin N-terminal peptide EDASSDSTGADPAQEAGTS (Pep Lac N-Term) were generated and characterized against human recombinant lacritin and N-65 truncation mutant. Basal tears were collected from 66 healthy individuals ranging in age from 18 to 52 years, and at four times during one 24-hour period from 34 other individuals. Lacritin levels were then analyzed by ELISA and Western blotting.

Results.

Anti-Pep Lac N-Term bound lacritin, but not truncation mutant N-65 that lacks the N-terminal antigenic site. Tear lacritin levels followed a normal distribution with a mean of 4.2 ± 1.17 ng/100 ng total tear protein. Levels differed little by age or sex, and decreased slightly between 4 and 8 hours in a 24-hour cycle. Tear-blocking effects were minimal, as suggested by spiking of tears with recombinant lacritin.

Conclusions.

Anti-Pep Lac N-Term–detectable lacritin comprises ∼4.2 ng/100 ng total tear protein in healthy individuals, with no significant differences between males and females or among individuals between 18 and 52 years old. Levels decrease slightly in the late afternoon. These findings provide a baseline for future immunodiagnostic studies of lacritin in dry eye and other ocular diseases.

Several studies suggest that lacritin may be selectively downregulated in dry eye and in blepharitis, yet little information is available about normal baseline levels. The results reported here indicate that lacritin comprises approximately 4.2 ng/100 ng total tear protein in healthy individuals with no significant differences between males and females, or by age between 18 and 52 years. The findings provide a baseline for future immunodiagnostic studies of lacritin in dry eye and other ocular diseases.

Purpose.

The ubiquitin–proteasome pathway (UPP) is an important protein quality control mechanism for selective degradation of abnormal proteins. The objective of this study was to test the hypothesis that enhancement of the UPP capacity could attenuate the accumulation and aggregation of misfolded proteins using V76D-γD-crystallin as a model substrate.

Methods.

Wild type (wt) and V76D mutant γD-crystallin were fused to red fluorescence protein (RFP) and expressed in human lens epithelial cells. The cellular distribution of the expressed proteins was compared by fluorescence microscopy. The solubility of wt- and V76D-γD-crystallin was determined by cellular fractionation and Western blotting. Wt-γD-RFP and V76D-γD-RFP were also cotransfected along with a ubiquitin ligase (CHIP) or a ubiquitin-conjugating enzyme (Ubc5) into cells. Levels of wt- and V76D-γD-crystallin, the percentage of transfected cells with aggregates, and aggregate size were quantified and compared among different groups.

Results.

Wt-γD-crystallin was evenly distributed in cells, whereas V76D-γD-crystallin formed intracellular aggregates. Eighty percent of wt-γD-crystallin was detected in the soluble fraction, whereas only 7% of V76D-γD-crystallin was soluble. CHIP or Ubc5 coexpression reduced the protein level of V76D-γD and concomitantly its aggregation in transfected cells; these effects could be attenuated by proteasome inhibitor. Mutant CHIP with defect TPR (tetratricopeptide repeat) or U-box domain failed to reduce levels of V76D-γD-crystallin.

Conclusions.

Enhancing ubiquitin conjugation activity reduces accumulation and aggregation of V76D-γD-crystallin by promoting its degradation. Upregulation of ubiquitin-conjugating activity could be an effective strategy to maintain lens transparency by eliminating other forms of misfolded proteins.

Enhancement of ubiquitin conjugation activity suppresses the aggregation of misfolded lens proteins in living cells by promoting their degradation.

Purpose.

To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells.

Methods.

MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot.

Results.

Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release.

Conclusions.

HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.

The study reports high glucose (HG) downregulates mitochondrial connexin 43 (mtCx43) expression and contributes to retinal vascular cell death. Additionally, HG reduces mtCx43 channel activity, induces mitochondrial fragmentation, and cytochrome c release resulting in retinal endothelial cell death. This study investigated the effects of altered mtCx43 expression on cellular dysfunction associated with demise of vascular cells in early stages of diabetic retinopathy. In particular, the findings indicate that HG-induced downregulation of mtCx43 contributes to mitochondrial shape change and subsequent cytochrome c release, which in turn, promotes apoptosis in retinal endothelial cells.

Purpose.

To document and explain the presence, inferior to the optic disc, of a distinct vertical boundary between two retinal areas of different short-wavelength autofluorescence (SW-AF) intensities.

Methods.

SW-AF images of the inferonasal region were acquired from 32 healthy subjects. Additionally, color, 488-nm reflectance (488-R), near-infrared reflectance (NIR-R), NIR autofluorescence (NIR-AF) images, and a spectral domain optical coherence tomography (SD-OCT) image were obtained in selected subjects. Gray levels (GL) on both sides of the demarcation line were measured in SW-AF and 488-R at fixed distances from the disc center.

Results.

A curved demarcation line inferior to the optic disc was observed on SW-AF images in 31/32 subjects. AF levels on the nasal side were 13% (±6%) lower than on the temporal side at 20° inferior to the disc center. The contrast between the nasal and the temporal areas was not significantly affected by age, sex, refractive error, race, or iris color. The demarcation line visible in SW-AF was also seen, though with reduced contrast, in approximately 80% of the 488-R images (lower reflectance on the nasal side) and 50% of color images. The boundary was not detected by NIR-R, NIR-AF, or by SD-OCT imaging.

Conclusions.

The location and the distinctness of the demarcation line may indicate a relationship to the closed embryonic optic fissure. The reduced SW-AF intensity and 488-R reflectance observed on the nasal side of this line may be attributable to lower lipofuscin and melanin content per unit area, possibly resulting from a difference in RPE cell shape.

In short-wavelength autofluorescence images a curved demarcation line can be observed inferior to the optic disc.

Purpose.

Thrombospondin-1 (TSP1) and TSP2 are matricellular proteins that have been shown to regulate cytoskeleton, cell adhesion, and extracellular matrix remodeling. Both TSP1 and TSP2 are found in the trabecular meshwork (TM). In cadaver eyes with primary open-angle glaucoma (POAG), TSP1 is increased in one third of patients. We hypothesized that TSP1 and TSP2 participate in the regulation of intraocular pressure (IOP).

Methods.

IOPs of TSP1-null, TSP2-null mice, and their corresponding wild-type (WT) mice were measured using a commercial rebound tonometer. Fluorophotometric measurements assessed aqueous turnover. Central corneal thickness (CCT) was measured by optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM).

Results.

Average IOPs of TSP1-null and TSP2-null mice were 10% and 7% less than that of the corresponding WT mice, respectively. CCTs were 6.5% less in TSP1-null mice (P < 0.05) and 1.1% less in TSP2-null mice (P > 0.05). Fluorophotometric measurements suggest that aqueous turnover rates in TSP1-null and TSP2-null mice are greater than those of WT mice. LM of the TSP1-null and TSP2-null iridocorneal angles reveals morphology, which is indistinguishable from that of their corresponding WTs. IF revealed possible concurrent underexpression of TSP2 in TSP1-null mice and of TSP1 in TSP2-null mice. TEM revealed larger collagen fibril diameters in TSP1-null and TSP2-null mice compared with WTs.

Conclusions.

TSP1-null and TSP2-null mice have lower IOPs than their WT counterparts. The rate of aqueous turnover suggests that the mechanism is enhanced outflow facility. An alteration in the extracellular matrix may contribute to this finding.

Thrombospondin-1 (TSP1) and TSP2 are matricellular proteins expressed in the trabecular meshwork. TSP1-null and TSP2-null mice have lower intraocular pressures than their wild-type counterparts. Aqueous humor turnover studies suggest that the mechanism is enhanced outflow resistance.

Purpose.

Stress of the endoplasmic reticulum and oxidative stress play critical roles in the pathogenesis of Fuchs Endothelial Corneal Dystrophy (FECD). In the normal aging cornea, cellular stress has been associated with a loss in proliferative capacity (premature senescence) of corneal endothelial cells (CECs). The present study used a transgenic Col8a2Q455K/Q455K knock-in mouse model of early-onset FECD to identify the endothelial expression profile of specific cellular stress response–related targets, which may be relevant to late-onset FECD.

Methods.

The differential endothelial mRNA levels of cellular stress response–related genes were determined in 12-month-old homozygous Col8a2Q455K/Q455K mutant and wild-type mice using customized PCR arrays. Result validation and analysis of additional senescence-related transcripts was performed by real-time PCR. Expression of p53 and p21 was assessed by immunofluorescence. Senescence-associated β-galactosidase (SA-β-Gal) activity was investigated by histochemical labeling. Human FECD samples and normal controls were examined for p21 expression by immunohistochemistry.

Results.

PCR-array analysis showed greater than 2-fold and/or significantly altered endothelial regulation of 19 cellular stress response-related transcripts in Col8a2Q455K/Q455K mutant mice; real-time PCR documented statistically significant upregulation of senescence-associated targets Cdkn1a (p21), Serpine1 (PAI-1), Tagln (Sm22), Fn1 and Clu (ApoJ). Immunofluorescence revealed increased expression of nuclear p53 and p21 in mutant animals. SA-β-Gal staining detected increased proportions of senescent CECs in mutant mice. Human FECD endothelium exhibited increased levels of nuclear p21 protein.

Conclusions.

Our results identify endothelial Cdkn1a (p21) upregulation in a mouse model of early-onset FECD, confirm overexpression of p21 in late-onset human FECD endothelium, and suggest a role for premature senescence in FECD.

Investigation of the corneal endothelium from a Fuchs endothelial corneal dystrophy (FECD) mouse model and from human FECD corneal samples provides first evidence for p21 overexpression and premature senescence in FECD.

Purpose.

We aim to understand how mechanical causation influences retinal detachment and reattachment processes. In particular, myopes suffer retinal detachment more frequently than emmetropes, and following a retinal detachment, scleral buckling promotes retinal reattachment. We test the hypothesis that stresses arising from saccadic eye rotations are involved in the processes, and that the alteration in the stress due to the change in the vitreous chamber geometry is sufficient to explain the phenomena.

Methods.

The vitreous chamber of the eye has an approximately spherical shape and it is filled with vitreous humor. We developed a mathematical model, treating the vitreous chamber in emmetropic and myopic eyes as a spheroid and in eyes subjected to scleral buckling as a sphere with a circumferential indentation. We assume that the eye performs prescribed small-amplitude, periodic, torsional rotations and we solve semi-analytically for the fluid pressure, velocity, and stress distributions.

Results.

The shape of the vitreous chamber has a large effect on the retinal stress. The vitreous and the retina of a highly myopic eye continuously experience shear stresses significantly higher than those of an emmetropic eye. An eye fitted with a scleral buckle experiences large stress levels localized around the buckle.

Conclusions.

Our results provide a mechanical explanation for the more frequent occurrence of posterior vitreous detachment and retinal detachment in myopic eyes. To understand how the stress distribution in a buckled eye facilitates reattachment, an additional model of the details of the reattachment process should be coupled to this model.

We present a mathematical model of the stress generated by the vitreous on the retina during eye movements. We investigate the role of the vitreous chamber shape on the stress distribution and intensity, focusing on myopic eyes and eyes subjected to scleral buckling.

Purpose.

To design and develop a drug-delivery system containing a combination of poly(d,l-lactide-co-glycolide) (PLGA) microparticles and alginate hydrogel for sustained release of retinoids to treat retinal blinding diseases that result from an inadequate supply of retinol and generation of 11-cis-retinal.

Methods.

To study drug release in vivo, either the drug-loaded microparticle–hydrogel combination was injected subcutaneously or drug-loaded microparticles were injected intravitreally into Lrat−/− mice. Orally administered 9-cis-retinoids were used for comparison and drug concentrations in plasma were determined by HPLC. Electroretinography (ERG) and both chemical and histologic analyses were used to evaluate drug effects on visual function and morphology.

Results.

Lrat−/− mice demonstrated sustained drug release from the microparticle/hydrogel combination that lasted 4 weeks after subcutaneous injection. Drug concentrations in plasma of the control group treated with the same oral dose rose to higher levels for 6−7 hours but then dropped markedly by 24 hours. Significantly increased ERG responses and a markedly improved retinal pigmented epithelium (RPE)–rod outer segment (ROS) interface were observed after subcutaneous injection of the drug-loaded delivery combination. Intravitreal injection of just 2% of the systemic dose of drug-loaded microparticles provided comparable therapeutic efficacy.

Conclusions.

Sustained release of therapeutic levels of 9-cis-retinoids was achieved in Lrat−/− mice by subcutaneous injection in a microparticle/hydrogel drug-delivery system. Both subcutaneous and intravitreal injections of drug-loaded microparticles into Lrat−/− mice improved visual function and retinal structure.

A novel drug-delivery system was developed for sustained release of therapeutic levels of 9-cis-retinoids. A PLGA microsphere alginate hydrogel combination was used both in vitro and in vivo to evaluate its therapeutic efficacy in retinas of Lrat−/− mice.