Showing papers in "Investigative Ophthalmology & Visual Science in 2005"

Macular Segmentation with Optical Coherence Tomography

Abstract: Purpose To develop a software algorithm to perform automated segmentation of retinal layer structures on linear macular optical coherence tomography (StratusOCT; Carl Zeiss Meditec, Inc., Dublin, CA) scan images and to test its performance in discriminating normal from glaucomatous eyes in comparison with conventional circumpapillary nerve fiber layer (cpNFL) thickness measurement.

Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration.

Abstract: PURPOSE Ranibizumab (rhuFab V2; Lucentis, Genentech, South San Francisco, CA) is a humanized monoclonal antibody fragment designed to bind all forms of VEGF, thereby blocking vessel permeability and angiogenesis in neovascular age-related macular degeneration. This study evaluated the pharmacokinetic (PK) and serum bioavailability of ranibizumab after a single intravitreal (ITV) or intravenous (IV) dose in cynomolgus monkeys. METHODS Monkeys received ranibizumab as either a bilateral ITV dose (500 or 2000 microg/eye; n = 6/group) or a single IV dose (1000 or 4000 microg/animal; n = 4/group). After ITV administration, ranibizumab concentrations were measured in several ocular compartments and in serum for 10 days and, after IV administration, for 48 hours. Pharmacokinetic parameters were estimated by compartmental and noncompartmental methods. RESULTS Ranibizumab cleared in parallel from all ocular compartments, with a terminal half-life of approximately 3 days. It distributed rapidly to the retina (6-24 hours), and concentrations were approximately one third that in the vitreous. After ITV injection, bioavailability (F) was 50% to 60%. Serum concentrations were very low, reflecting wider distribution and faster clearance when ranibizumab reached the serum. After IV administration, the terminal half-life was approximately 0.5 day. CONCLUSIONS This study demonstrates that ranibizumab has a PK profile that is favorable for its clinical use in treating neovascular AMD by monthly ITV injection.

Factors influencing optic nerve head biomechanics.

Abstract: measures. RESULTS. The five input factors that had the largest influence across all outcome measures were, in ranked order: stiffness of the sclera, radius of the eye, stiffness of the lamina cribrosa, IOP, and thickness of the scleral shell. The five least influential factors were, in reverse ranked order: retinal thickness, peripapillary rim height, cup depth, cup-to-disc ratio, and pial thickness. Factor ranks were similar for various outcome measure groups and factor ranges. CONCLUSIONS. The model predicts that ONH biomechanics are strongly dependent on scleral biomechanical properties. Acute deformations of ONH tissues, and the consequent high levels of neural tissue strain, were less strongly dependent on the action of IOP directly on the internal surface of the ONH than on the indirect effects of IOP on the sclera. This suggests that interindividual variations in scleral properties could be a risk factor for the development of glaucoma. Eye size and lamina cribrosa biomechanical properties also have a strong influence on ONH biomechanics. (Invest Ophthalmol Vis Sci. 2005;46: 4189‐4199) DOI:10.1167/iovs.05-0541

The Ins2Akita mouse as a model of early retinal complications in diabetes.

Abstract: PURPOSE This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. METHODS Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. RESULTS The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia. CONCLUSIONS The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.

Changes in collagen orientation and distribution in keratoconus corneas

Abstract: PURPOSE. To map the collagen orientation and relative distribution of collagen fibrillar mass in keratoconus corneal buttons. METHODS. Structural analysis was performed by obtaining synchrotron x-ray scattering patterns across the samples at 0.25-mm intervals. The patterns were analyzed to produce two-dimensional maps of the orientation of the lamellae and of the distribution of total and preferentially aligned lamellae. RESULTS. Compared with normal corneas, in keratoconus the gross organization of the stromal lamellae was dramatically changed, and the collagen fibrillar mass was unevenly distributed, particularly around the presumed apex of the cone. CONCLUSIONS. The development of keratoconus involves a high degree of inter- and probably intralamellar displacement and slippage that leads to thinning of the central cornea and associated changes in corneal curvature. This slippage may be promoted by a loss of cohesive forces and mechanical failure in regions where lamellae bifurcate.

Prevalence of diabetic retinopathy in urban India: the Chennai Urban Rural Epidemiology Study (CURES) eye study, I.

Abstract: PURPOSE. To assess the prevalence of diabetic retinopathy (DR) in type 2 diabetic subjects in urban India using four-field stereo color photography. METHODS. The Chennai Urban Rural Epidemiology Study (CURES) is a population-based study conducted on a representative population of Chennai (formerly Madras) city in South India. Individuals 20 years in age (n 26,001) were screened for diabetes. Of the 1529 known diabetic subjects, 1382 (90.4%) participated in the study. Subjects with newly detected diabetes (n 354) by the oral glucose tolerance test (OGTT) also consented to participate in the study. All the subjects underwent four-field stereo color photography, and retinopathy was graded in the color fundus photographs according to Early Treatment Diabetic Retinopathy Study (ETDRS) criteria. RESULTS. The overall prevalence of DR in the population was 17.6% (95% confidence interval [CI]: 15.8 –19.5), which included 20.8% (95% CI: 18.7–23.1) in known diabetic subjects and 5.1% (95% CI: 3.1– 8.0) in subjects with newly detected diabetes. The prevalence of DR was significantly higher in men than in women (21.3% vs. 14.6%; P 0.0001) and among subjects with proteinuria (P 0.002). Logistic regression analysis showed that for every 5-year increase in the duration of diabetes, the risk for DR increased 1.89-fold (95% CI: 1.679– 2.135; P 0.0001). For every 2% elevation of glycated hemoglobin (HbA1c), the risk for DR increased by a factor of 1.7 (95% CI: 1.545–1.980; P 0.0001). CONCLUSIONS. This study shows that the prevalence of diabetic retinopathy is lower in urban South Indians than in other ethnic groups. However, due to the large number of diabetic subjects, DR is likely to pose a public health burden in India; hence, routine retinal examination is mandatory to detect DR in the early stages.

RPE65 Is the Isomerohydrolase in the Retinoid Visual Cycle

Abstract: RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol/min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.

Peripheral vision can influence eye growth and refractive development in infant monkeys.

Abstract: PURPOSE—Given the prominence of central vision in humans, it has been assumed that visual signals from the fovea dominate emmetropization. The purpose of this study was to examine the impact of peripheral vision on emmetropization. METHODS—Bilateral, peripheral form deprivation was produced in 12 infant monkeys by rearing them with diffusers that had either 4- or 8-mm apertures centered on the pupils of each eye, to allow 24° or 37° of unrestricted central vision, respectively. At the end of the lens-rearing period, an argon laser was used to ablate the fovea in one eye of each of seven monkeys. Subsequently, all the animals were allowed unrestricted vision. Refractive error and axial dimensions were measured along the pupillary axis by retinoscopy and A-scan ultrasonography, respectively. Control data were obtained from 21 normal monkeys and 3 infants reared with binocular plano lenses. RESULTS—Nine of the 12 treated monkeys had refractive errors that fell outside the 10th- and 90th-percentile limits for the age-matched control subjects, and the average refractive error for the treated animals was more variable and significantly less hyperopic/more myopic (+0.03 ± 2.39 D vs. +2.39 ± 0.92 D). The refractive changes were symmetric in the two eyes of a given animal and axial in nature. After lens removal, all the treated monkeys recovered from the induced refractive errors. No interocular differences in the recovery process were observed in the animals with monocular foveal lesions.

Incidence and progression of myopia in Singaporean school children.

Abstract: Purpose To determine the incidence and progression rates of myopia in young Singaporean children. Methods A prospective cohort study, the Singapore Cohort Study of the Risk Factors for Myopia (SCORM), was conducted in two schools in Singapore (1999-2002). Children aged 7 to 9 years (n=981) were followed up over a 3-year period. Cycloplegic autorefraction and biometry parameter measures were performed annually, according to the same protocol. Results The 3-year cumulative incidence rates were 47.7% (95% confidence interval [CI]: 42.2-53.3), 38.4% (95% CI: 31.4-45.4), and 32.4% (95% CI: 21.8-43.1) for 7-, 8-, and 9-year-old children, respectively. The 3-year cumulative incidence rates were higher in Chinese (49.5% vs. 27.2%) and in 7-year-old compared with 9-year-old children at baseline (47.7% vs. 32.4%), though the latter relationship was of borderline significance after adjustment for race, gender, amount of reading (books/week), and parental myopia (P=0.057). Premyopic children with greater axial lengths, vitreous chamber depths, and thinner lenses were more prone to the development of myopia, after controlling for age, gender, race, reading, and parental myopia. The 3-year mean cumulative myopia progression rates were -2.40 D (95% CI: -2.57 to -2.22) in 7-year-old myopic children, -1.97 (95% CI: -2.16 to -1.78) in 8-year-olds, and -1.71 (95% CI: -1.98 to -1.44) in 9-year-olds. Conclusions Both the incidence and progression rates of myopia are high in Singaporean children.

Fate maps of neural crest and mesoderm in the mammalian eye

Abstract: PURPOSE. Structures derived from periocular mesenchyme arise by complex interactions between neural crest and mesoderm. Defects in development or function of structures derived from periocular mesenchyme result in debilitating vision loss, including glaucoma. The determination of long-term fates for neural crest and mesoderm in mammals has been inhibited by the lack of suitable marking systems. In the present study, the first long-term fate maps are presented for neural crest and mesoderm in a mammalian eye. METHODS. Complementary binary genetic approaches were used to mark indelibly the neural crest and mesoderm in the developing eye. Component one is a transgene expressing Cre recombinase under the control of an appropriate tissue-specific promoter. The second component is the conditional Cre reporter R26R, which is activated by the Cre recombinase expressed from the transgene. Lineage-marked cells were counterstained for expression of key transcription factors. RESULTS. The results established that fates of neural crest and mesoderm in mice were similar to but not identical with those in birds. They also showed that five early transcription factor genes are expressed in unique patterns in fate-marked neural crest and mesoderm during early ocular development. CONCLUSIONS. The data provide essential new information toward understanding the complex interactions required for normal development and function of the mammalian eye. The results also underscore the importance of confirming neural crest and mesoderm fates in a model mammalian system. The complementary systems used in this study should be useful for studying the respective cell fates in other organ systems.

Retinal Ganglion Cell Apoptosis in Glaucoma Is Related to Intraocular Pressure and IOP-Induced Effects on Extracellular Matrix

Abstract: Glaucoma is a major cause of worldwide irreversible blindness. Vision loss in glaucoma is attributed to retinal ganglion cell (RGC) death, and intraocular pressure (IOP) is the major modifiable risk factor.1 The underlying mechanisms that link elevated IOP to glaucomatous RGC death are not fully understood, though the process of RGC apoptosis is heavily implicated.2-4 Furthermore, the site of primary damage in glaucoma is controversial, although it is suspected to be either at the optic nerve head (ONH) or the retina and is complicated by the likelihood of the effects of secondary RGC degeneration.5 An established theory is that elevated IOP induces physical changes at the ONH, visualized clinically as optic disc cupping, which causes optic nerve axonal compression at the lamina cribrosa, blockage of axoplasmic flow, and interference in retrograde neurotrophin transport to RGCs, leading to cell death. The ONH has previously been investigated in both human and experimental animal models as a primary site of glaucomatous damage. These studies have shown extensive remodeling of the extracellular matrix (ECM), including collagen I and IV, TGF-β2, and matrix metalloproteinase (MMP)-1.6-13 Hernandez8 has identified the astrocytes as the key cell type involved in this process at the ONH and has shown astrocytes to be activated by increased IOP. Yan et al.9 have shown that these active astrocytes are responsible for the production of the matrix-degrading enzymes (MMPs) that affect the pattern of matrix remodeling.10 It is believed that these effects are modulated by astrocyte production of TGF-β2, which has been shown to be significantly increased in the glaucomatous ONH.12 Previous studies have shown a positive correlation of the loss of RGC axons to the level and the duration of IOP elevation in experimental rat glaucoma.14,15 It is estimated that approximately half of the total RGCs are lost after 2 to 3 months of IOP elevation.16-18 However, there has been little research on the direct effect of IOP on RGC apoptosis, although recent in vitro studies suggest RGC apoptosis may be induced directly by elevated IOP, supporting the theory that the retina is the primary injury site in glaucoma.19 The mechanisms by which RGC apoptosis occurs has recently been linked to specific ECM-related changes in MMP-9 and laminin expression in the retina.20,21 This is further corroborated by the fact that in the central nervous system (CNS), neuronal apoptosis is associated with increased MMP-9 activity.22,23 We have investigated for the first time, as far as we are aware, the effect of IOP elevation on the level of RGC apoptosis in an in vivo model of glaucoma. As RGC apoptosis has been recently linked to specific ECM changes,20,21 we have evaluated the role of MMP-9, laminin, and TIMP-1 activity in the retina, and whether RGC apoptosis is at all related to IOP-induced effects on these targets. In addition, we have assessed the effects of IOP on changes in collagen I, IV, MMP-1, and TGF-β2 at both the ONH and retina.

Reproducibility of retinal nerve fiber thickness measurements using the stratus OCT in normal and glaucomatous eyes.

Abstract: PURPOSE. To determine the reproducibility of Stratus Optical Coherence Tomography (OCT) retinal nerve fiber layer (RNFL) measurements around the optic nerve in normal and glaucomatous eyes. METHODS. One eye was chosen at random from 88 normal subjects and 59 glaucomatous subjects distributed among mild, moderate, and severe glaucoma, determined by visual field testing. Subjects underwent six RNFL thickness measurements performed by a single operator over a 30-minute period with a brief rest between sessions. Three scans were taken with the high-density Standard RNFL protocol, and three were taken with the Fast RNFL protocol, alternating between scan protocols. RESULTS. Reliability, as measured by intraclass correlation coefficient (ICC), was calculated for the overall mean RNFL thickness and for each quadrant. The ICC for the mean Standard RNFL thickness (and lower 95% confidence interval [CI]) in normal and glaucomatous eyes was 0.97 (0.96 CI) and 0.98 (0.97 CI), respectively. The ICC for the mean Fast RNFL thickness in normal and glaucomatous eyes was 0.95 (0.93 CI) and 0.97 (0.95 CI), respectively. Quadrant ICCs ranged between 0.79 and 0.97, with the nasal quadrant being the least reproducible of all four quadrants, using either the Standard or Fast RNFL program. The test‐retest variability ranged from 3.5 m for the average RNFL thickness measurements in normal eyes to 13.8 m for the nasal quadrant measurements in glaucomatous eyes, which appeared to be the most variable. CONCLUSIONS. Reproducibility of RNFL measurements using the Stratus OCT is excellent in normal and glaucomatous eyes. The nasal quadrant appears to be the most variable measurement. Standard RNFL and Fast RNFL scans are equally reproducible and yield comparable measurements. These findings have im

Axial growth and changes in lenticular and corneal power during emmetropization in infants.

Abstract: PURPOSE To evaluate the contribution made by the ocular components to the emmetropization of spherical equivalent refractive error in human infants between 3 and 9 months of age. METHODS Keratophakometry in two meridians was performed on 222 normal-birthweight infant subjects at 3 and 9 months of age. The spherical equivalent refractive error was measured by cycloplegic retinoscopy (cyclopentolate 1%). Anterior chamber depth, lens thickness, and vitreous chamber depth were measured by A-scan ultrasonography over the closed eyelid. RESULTS Both the mean and SD for spherical equivalent refractive error decreased between 3 and 9 months of age (+2.16 +/- 1.30 D at 3 months; +1.36 +/- 1.06 D at 9 months; P < 0.0001, for the change in both mean and SD). Average ocular component change was characterized by increases in axial length, thinning, and flattening of the crystalline lens, increases in lens equivalent refractive index, and decreases in lens and corneal power. Initial refractive error was associated in a nonlinear manner with the change in refractive error (R(2) = 0.41; P < 0.0001) and with axial growth (R(2) = 0.082; P = 0.0005). Reduction in hyperopia correlated significantly with increases in axial length (R(2) = 0.16; P < 0.0001), but not with changes in corneal and lenticular power. Decreases in lenticular and corneal power were associated with axial elongation (R(2) = 0.40, R(2) = 0.12, respectively; both P < 0.0001). CONCLUSIONS Modulation in the amount of axial growth in relation to initial refractive error appeared to be the most influential factor in emmetropization of spherical equivalent refractive error. The associations between initial refractive error, subsequent axial growth, and change in refractive error were consistent with a visual basis for emmetropization. The cornea and crystalline lens lost substantial amounts of dioptric power in this phase of growth, but neither appeared to play a significant role in emmetropization.

Curcumin and turmeric delay streptozotocin-induced diabetic cataract in rats.

Abstract: Purpose The purpose of this study was to investigate the effect of curcumin and its source, turmeric, on streptozotocin-induced diabetic cataract in rats. Methods Wistar-NIN rats were selected and diabetes was induced by streptozotocin (35 mg/kg body weight, intraperitoneally) and divided into four groups (group II-V). The control (group I) rats received only vehicle. Group I and II animals received an unsupplemented AIN-93 diet, and those in groups III, IV, and V received 0.002% and 0.01% curcumin and 0.5% turmeric, respectively, in an AIN-93 diet for a period of 8 weeks. Cataract progression due to hyperglycemia was monitored by slit lamp biomicroscope and classified into four stages. At the end of 8 weeks, the animals were killed and the biochemical pathways involved in the pathogenesis of cataract such as oxidative stress, polyol pathway, alterations in protein content and crystallin profile in the lens were investigated, to understand the possible mechanism of action of curcumin and turmeric. Blood glucose and insulin levels were also determined. Results Although, both curcumin and turmeric did not prevent streptozotocin-induced hyperglycemia, as assessed by blood glucose and insulin levels, slit lamp microscope observations indicated that these supplements delayed the progression and maturation of cataract. The present studies suggest that curcumin and turmeric treatment appear to have countered the hyperglycemia-induced oxidative stress, because there was a reversal of changes with respect to lipid peroxidation, reduced glutathione, protein carbonyl content and activities of antioxidant enzymes in a significant manner. Also, treatment with turmeric or curcumin appears to have minimized osmotic stress, as assessed by polyol pathway enzymes. Most important, aggregation and insolubilization of lens proteins due to hyperglycemia was prevented by turmeric and curcumin. Turmeric was more effective than its corresponding levels of curcumin. Conclusions The results indicate that turmeric and curcumin are effective against the development of diabetic cataract in rats. Further, these results imply that ingredients in the study's dietary sources, such as turmeric, may be explored for anticataractogenic agents that prevent or delay the development of cataract.

Reduced foveolar choroidal blood flow in eyes with increasing AMD severity.

Abstract: PURPOSE. In an earlier study, the authors reported that foveolar choroidal blood flow (ChBFlow) decreases in patients with AMD and drusen. To explore further the choroidal circulatory changes in patients with AMD, the relationship between ChBFlow and fundus features associated with increased risk of choroidal neovascularization (CNV) were investigated. METHODS. The study included 26 control eyes of 17 normal subjects and 163 eyes with early AMD characteristics of 123 patients with AMD. The AMD study eyes were divided into three groups according to increasing risk for development of CNV: (1) drusen 63 m, no RPE hyperpigmentary changes in the study eye, and no CNV in the fellow eye; (2) drusen 63 m, RPE hyperpigmentary changes in the study eye, and no CNV in the fellow eye; and (3) eyes with CNV in the fellow eye. Laser Doppler flowmetry was used to assess relative foveolar choroidal blood velocity (ChBVel), volume (ChBVol), and flow (ChBFlow). Differences in the mean circulatory parameters were assessed by analysis of variance (ANOVA) and test of linear trend. RESULTS. Mean ChBVel, ChBVol, and ChBFlow decreased with increased risk for CNV (linear trend, P 0.05). The lowest circulatory parameters were observed in the eyes with the highest risk for CNV development. Trends for ChBVel and ChBFlow were still significant after adjustment for multiple factors. CONCLUSIONS. There is a systematic decrease in choroidal circulatory parameters with an increase in the severity of AMD features associated with risk for the development of CNV, suggesting a role for ischemia in the development of CNV. (Invest Ophthalmol Vis Sci. 2005;46:1033‐1038) DOI:10.1167/ iovs.04-1050

Ocular Rigidity in Living Human Eyes

Abstract: PURPOSE. To measure the rigidity coefficient of a large number of subjects at clinically encountered intraocular pressures (IOPs) and to examine the possible correlation of ocular rigidity with other factors, such as the age of the patients, ocular parameters (axial length and corneal thickness), and pathologic conditions affecting the eye. METHODS. The pressure‐volume relationship and the ocular rigidity coefficient (K) were determined in 79 eyes undergoing cataract surgery, by injecting 200 L of saline solution (in steps of 4.5 L) through the limbus into the anterior chamber, while continually monitoring the IOP with a transducer, up to the limit of 60 mm Hg. Data within an IOP range of 10 to 35 mm Hg were used to calculate the scleral rigidity coefficient. All measurements were taken at the same time of day, to eliminate any possible diurnal variation. RESULTS. The mean ocular rigidity coefficient was 0.0126 mm Hg/L (95% confidence interval [CI], 0.0112‐0.0149). A statistically significant positive correlation between the rigidity coefficient and age of the patient was found (P 0.02), whereas similar findings were not observed for the examined ocular parameters (axial length, P 0.09; and corneal thickness, P 0.12). No correlation was found for patients with diabetes mellitus (P 0.39), age-related macular degeneration (P 0.55), and hypertension (P 0.45). CONCLUSIONS. The present study provides quantitative data on the ocular rigidity coefficient based on measurements in a large series of living human eyes. A positive correlation between the ocular rigidity coefficient and the patient’s age was documented. (Invest Ophthalmol Vis Sci. 2005;45:409‐414)

Three-dimensional ultrahigh-resolution optical coherence tomography of macular diseases.

Abstract: PURPOSE. To demonstrate a new generation of three-dimensional (3-D) ultrahigh-resolution optical coherence tomography (UHR OCT) technology for visualization of macular diseases. METHODS. One hundred forty eyes with a distinct disease in each of the posterior pole compartments were examined with 3-D UHR OCT. 3-D imaging was performed with a high axial resolution of 3 μm with a compact, commercially available, ultra-broad-bandwidth (160 nm) titanium:sapphire laser at a video rate of up to 25 B-scans/s. Each tomogram consisted of 1024 X 1024 pixels, resulting in 25 megavoxels/s. RESULTS. 3-D UHR OCT offers high-precision 3-D visualization of macular diseases at all structural levels. The UHR modality allows identification of the contour of the hyaloid membrane, tractive forces of epiretinal membranes, and changes within the inner limiting membrane. The system provides quality 3-D images of the topographic dynamics of traction lines from the retinal surface down to the level of the photoreceptor segments. Intraretinal diseases are identified by their specific location in different layers of the neurosensory ultrastructure. Photoreceptor inner and outer segments are clearly delineated in configuration and size, with a characteristic peak in the subfoveal area. The microarchitecture of choroidal neovascularization is distinctly imaged, related leakage can be identified, and the volume can be quantified. CONCLUSIONS. High-speed UHR OCT offers unprecedented, realistic, 3-D imaging of ocular diseases at all epi-, intra- and subretinal levels. A complete 3-D data set of the macular layers allows a comprehensive analysis of focal and diffuse diseases, as well as identification of dynamic pathomechanisms.

Immunosuppressive factors secreted by human amniotic epithelial cells.

Abstract: PURPOSE. Amniotic membrane has been applied to the ocular surface to restore corneal function. The beneficial effect of amniotic membrane transplantation may be due to the immunosuppressive effects of amniotic epithelial cells. The purpose of this study was to determine whether amniotic epithelial cells (AECs) secrete anti-inflammatory and antiproliferative factors that affect the chemotaxis of neutrophils and macrophages and suppress both T- and B-cell proliferation in vitro. METHODS. Human amniotic cells were isolated from human amniotic membrane and cultured in vitro. The supernatants from AEC cultures were collected after 48 hours of incubation. Neutrophil and macrophage chemotactic activity was tested in the presence of AEC supernatant, using 24-well migration assay chambers. Lymphocyte proliferation was tested by H 3 -thymidine incorporation. Apoptosis was examined by caspase-3 and annexin V assays, and expression of cytokines was assessed by RT-PCR. RESULTS. AEC supernatant significantly inhibited the chemotactic activity of neutrophils and macrophages toward macrophage inflammatory protein (MIP)-2 (P 0.05). The supernatant significantly reduced the proliferation of both T and B cells after mitogenic stimulation (P 0.05). Caspase-3 assays revealed that the supernatant induced apoptosis of T and B cells, but not of corneal epithelial cells and liver cells. In contrast to lymphocytes, macrophages and neutrophils were resistant to apoptosis induced by AEC supernatant. The AECs expressed message for TNF, Fas ligand (FasL), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), TGF, and macrophage migration-inhibitory factor (MIF). However, AEC induction of apoptosis was inhibited (50%) by anti-FasL antibody but not by anti-TRAIL or anti-TNF antibodies. Moreover, AEC supernatant inhibited macrophage migration in vitro. CONCLUSIONS. AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems. (Invest Ophthalmol Vis Sci. 2005;46:900‐907) DOI:10.1167/iovs.04-0495

Design of a High–Resolution Optoelectronic Retinal Prosthesis

Abstract: It has been demonstrated that electrical stimulation of the retina can produce visual percepts in blind patients suffering from macular degeneration and retinitis pigmentosa. However, current retinal implants provide very low resolution (just a few electrodes), whereas at least several thousand pixels would be required for functional restoration of sight. This paper presents the design of an optoelectronic retinal prosthetic system with a stimulating pixel density of up to 2500 pix mm(-2) (corresponding geometrically to a maximum visual acuity of 20/80). Requirements on proximity of neural cells to the stimulation electrodes are described as a function of the desired resolution. Two basic geometries of sub-retinal implants providing required proximity are presented: perforated membranes and protruding electrode arrays. To provide for natural eye scanning of the scene, rather than scanning with a head-mounted camera, the system operates similar to 'virtual reality' devices. An image from a video camera is projected by a goggle-mounted collimated infrared LED-LCD display onto the retina, activating an array of powered photodiodes in the retinal implant. The goggles are transparent to visible light, thus allowing for the simultaneous use of remaining natural vision along with prosthetic stimulation. Optical delivery of visual information to the implant allows for real-time image processing adjustable to retinal architecture, as well as flexible control of image processing algorithms and stimulation parameters.

Mapping of the normal human corneal sub-Basal nerve plexus by in vivo laser scanning confocal microscopy.

Abstract: Purpose To produce a two-dimensional reconstruction map of the living human sub-basal corneal nerve plexus using in vivo confocal microscopy. Methods Laser scanning in vivo confocal microscopy was performed on three normal eyes of three healthy human subjects. Subjects were asked to fixate on targets arranged in a grid to enable examination of the cornea in a wide range of positions. Using the section mode, a mean of 573 +/- 176 images of the sub-basal plexus were obtained for each subject. The data were arranged and images were mapped for each subject into confluent montages. Results Mean dimensions of the corneal areas mapped were 4.95 +/- 0.53 mm horizontally and 5.14 +/- 0.53 mm vertically. In all subjects, the sub-basal nerve plexus appeared to radiate toward a whorl-like complex centered 1 to 2 mm inferior to the corneal apex. Outside this area, the nerve fiber bundles in the remainder of the cornea were arranged in a radiating pattern. Mean nerve density was significantly higher in the inferocentral whorl region (25,249 +/- 616 microm/mm2) compared with the central cornea (21,668 +/- 1411 microm/mm2) (Mann-Whitney U test; P = 0.05). Conclusions This is the first study to elucidate the overall distribution of sub-basal nerves in the healthy, live central to mid-peripheral human cornea by laser scanning in vivo confocal microscopy. The whorl pattern of the sub-basal nerves is similar to that seen in the epithelium in corneal verticillata and may lend support to the theory that epithelial cells and nerves migrate centripetally in tandem.

Serum inflammatory markers in diabetic retinopathy.

Abstract: PURPOSE To evaluate the association of serum factors with the severity of diabetic retinopathy and to assess their presence in retinal tissue obtained at autopsy. METHODS The following serum factors of 93 subjects were examined at the National Eye Institute (NEI) clinical center: the chemokines regulated on activation, normal T-cell expressed and presumably secreted (RANTES)/CCL5, epithelial neutrophil activator (ENA)-78/CXCL5, interferon-induced protein (IP)-10/CXCL10, stromal cell-derived factor (SDF)-1alpha/CXCLl2, monocyte chemoattractant protein (MCP)-1/CCL2, macrophage inflammatory protein (MIP)-1alpha/CCL3, interleukin (IL)-8/CXCL8; the cytokine IL-6; the cell adhesion molecules intercellular adhesion molecule (ICAM-1/CD54) and vascular cell adhesion molecule (VCAM/CD106); and the growth factor vascular endothelial growth factor (VEGF). Logistic regression was performed to assess the association of these factors with age, sex, severity of retinopathy, hemoglobin A(1C), total cholesterol, creatinine, duration of diabetes, and presence of macular edema. The outcome assessed was severity of retinopathy. Frozen sections of two donor eyes obtained at autopsy from a donor with documented severe nonproliferative diabetic retinopathy and diabetic macular edema and of a normal nondiabetic eye were processed by immunoperoxidase staining with primary antibodies against RANTES, MCP-1, ICAM-1, and LFA-1alpha/CD11a. RESULTS The levels of RANTES and SDF-1alpha were significantly elevated in patients with at least severe nonproliferative diabetic retinopathy compared with those with less severe diabetic retinopathy (P < 0.001 and 0.007, respectively). Positive immunostaining was observed in the inner retina for MCP-1 and RANTES of the patient with diabetes. Staining was strongly positive throughout the diabetic retina for ICAM-1. Normal retinal tissues showed little reactivity. CONCLUSIONS Serum chemokines were significantly elevated in patients with at least severe nonproliferative diabetic retinopathy compared with those who had less severe retinopathy. Elevated levels of the chemokines and cell adhesion molecules were also identified in eyes of a donor with ischemic diabetic retinopathy. These findings provide evidence to support the role of inflammation in the pathogenesis of diabetic retinopathy.

Vascular Damage in a Mouse Model of Diabetic Retinopathy: Relation to Neuronal and Glial Changes

Abstract: Purpose Lack of information about the development of diabetic retinopathy in mice has greatly hindered the use of genetic mouse models for the study of disease mechanisms and the development of therapeutic strategies. The objective of this study was to characterize the occurrence and pathologic progression of diabetic retinopathy in C57Bl/6J mice. Methods Diabetes was induced with five consecutive injections of streptozotocin (STZ). The retinas were collected at different time points (2 weeks to 22 months) after the induction of diabetes and examined by using molecular, histologic, and immunohistochemical techniques and morphometric analysis. Results There was transient induction of cell apoptosis and caspase-3 activation in retinal neurons of C57Bl/6 mice within days of diabetes induction. Glial fibrillary acidic protein (GFAP), a marker of glial activation, likewise was transiently upregulated, seemingly in astrocytes but not in Muller cells. These abnormalities quickly returned to normal; ultimately, no detectable loss of retinal ganglion cells (RGCs) was noted by any of three independent methods (number of cells in ganglion cell layer of retinal cross-sections, retrograde labeling of retinal ganglion cells with fluorescent dye, or TUNEL staining) after up to a 1-year duration of diabetes. Despite this apparent lack of evidence for progressive damage in neurons and glial cells, diabetic mice developed vascular disease characteristic of the early stage of diabetic retinopathy beginning at 6 months after the onset of disease. The vascular damage-formation of acellular capillaries and pericyte ghosts-continued to increase through the 18 months examined. Conclusions Diabetic C57Bl/6J mice develop capillary lesion that are characteristic of the early stages of diabetic retinopathy in patients. The data suggest that diabetes-induced degeneration of retinal capillaries can develop independent of neuronal loss or chronic GFAP upregulation in glial cells.

Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.

Abstract: PURPOSE. To develop and characterize a telomerase-immortalized human corneal epithelial cell line (hTCEpi) to serve as an in vitro model for studying the molecular mechanisms involved in regulating human corneal epithelial cell differentiation. METHODS. Primary cultures of human corneal epithelial cells were infected with a retroviral vector encoding human telomerase reverse transcriptase (hTERT). Infected hTCEpi cells were selected, cloned, and characterized to identify telomerase activity, proliferative capacity, karyotype, and differentiative potential in routine culture and under consecutive submerged and air-lifted conditions. Cells were evaluated to measure cell cycle kinetics (anti-Ki-67, anti-p16), stratification (phalloidin and anti-ZO-1), and differentiation (anti-K3, anti-BCL-2 and TUNEL labeling). RESULTS. hTCEpi cells exhibited telomerase activity, a normal karyotype and cell cycle kinetics at greater than 240 population doublings, and loss of p16 after passage 10. Air-lifting produced a well stratified epithelium (five to seven cell layers) with apical ZO-1-stained tight junctions. Submersed culture demonstrated increasing expression of stratification markers (K5/K14) with K3-corneal keratin marker expression in longterm, air-lifted culture. Anti-BCL-2 staining showed both nuclear and cytoplasmic localization with loss of nuclear BCL-2 expression in TUNEL-labeled surface epithelial cells. CONCLUSIONS. hTCEpi cells stratify, differentiate, and desquamate similar to normal human corneal epithelium. Further study of the hTCEpi cell line may be valuable in studying the molecular mechanisms regulating corneal epithelial cell differentiation and desquamation. (Invest Ophthalmol Vis Sci. 2005; 46:470‐478) DOI:10.1167/iovs.04-0528

HSV-1 DNA in tears and saliva of normal adults

Abstract: Healthy individuals are known to shed herpesviruses. Humans are reservoirs, and asymptomatic shedding is a major factor in the spread of the virus. In a recent 14-month study, Epstein-Barr virus (EBV) was present in all 30 individuals tested, and 4 (13%) of 30 shed cytomegalovirus (CMV) in urine.1 In a cross-sectional study in Eritrea, Africa, with the exception of children 80% herpes simplex virus type 1 (HSV-1) seropositive, and >90% CMV and varicella zoster virus (VZV) seropositive.2 Antibodies for HSV-1 have also been detected in tears (73%) and saliva (2.5%) of 40 healthy individuals.3 HSV-1 and, to a lesser extent, HSV-2 are known to be the leading causes of virus-induced blindness in the Western world, with approximately 500,000 individuals having herpetic eye disease in the United States.4 More than 62% of the U.S. population >12 years of age is positive for HSV-1, HSV-2, or both.4 Worldwide, 60% to 90% of the adult population is HSV-1 antibody positive.5,6 In one study, 100% of individuals older than 60 years were found to be HSV-1 seropositive.7 Despite the prevalence of HSV infections, however, only a small number of latently infected humans experience symptomatic disease. Only 1% to 6% of primary infections are clinically recognized.8 Consequently, asymptomatic shedding of HSV is considered the major form of transmission. Thus far, studies have focused on quantifying HSV-1 and -2 antibody titers and/or frequency of shedding in patients with active herpesvirus lesions or immediately after the time of active lesions.9 Although HSV-1 seropositivity in healthy individuals has also been evaluated,3,10 asymptomatic shedding of HSV-1, especially in tears, has received less attention. To the best of our knowledge, only three studies have evaluated HSV-1 shedding in tears and saliva of healthy individuals, using relatively insensitive culturing techniques.11–13 Detection of HSV shedding is dependent on the population surveyed, as well as on the diagnostic technique used. Before the advent of real-time PCR, the presence of HSV could be determined only by relatively insensitive culturing of infectious virus.14–21 Today we are able to detect HSV DNA with PCR, the new gold standard for HSV detection in clinical samples. Hence, the percentage of individuals detected as positive for HSV-1 DNA is expected to increase significantly, as has the percentage of individuals shedding EBV in saliva.1 To the best of our knowledge, the present study is the largest cross-sectional assessment performed to date of the presence of HSV-1 DNA in the eyes and mouths of healthy individuals, in terms of population size and total samples collected. We used real-time PCR to quantify the presence and frequency of asymptomatic viral shedding in a combined total of 5529 tear and saliva specimens. Samples consisted of mouth and eye swabs collected twice daily from 50 volunteers over the course of 30 days. The subjects were instructed to swab the mouth and one eye morning and night. In addition, serum HSV IgG antibody titers were determined. We found that 98% (49/50) of the subjects in our study were positive at least once for HSV-1 DNA in tears and/or saliva.

Expression of acute-phase response proteins in retinal Müller cells in diabetes.

Abstract: Purpose To characterize the whole spectrum of gene expression changes induced by diabetes in retinal Muller glial cells. Methods Muller cells were isolated from the retina of streptozotocin-diabetic and age-matched control rats by gradient centrifugation and immediately processed for RNA isolation. The gene expression profile of Muller cells was studied with the GeneChip Rat Genome oligonucleotide array (Affymetrix, Santa Clara, CA). The upregulation of acute-phase proteins in the retina of diabetic rats was confirmed by Northern and Western blot analyses. Real-time-RT-PCR was used to study the retinal expression of inflammatory cytokines. Results Gene expression profiling identified 78 genes as differentially expressed in diabetic Muller cells. One third of these genes were associated with inflammation, including a large cluster (18% of the differentially expressed genes) of acute-phase response proteins: alpha2-macroglobulin, ceruloplasmin, complement components, lipocalin-2, metallothionein, serine protease inhibitor-2, transferrin, tissue inhibitor of metalloproteases-1, transthyretin, and the transcription factor C/EBPdelta. Northern and Western blot analyses confirmed the upregulation of alpha2-macroglobulin and ceruloplasmin in the diabetic retina, but not in the cerebral cortex and liver of the same animals. The acute-phase response of Muller cells in diabetes was associated with upregulation of interleukin (IL)-1beta in the retina. Conclusions Muller cells acquire a complex and specific reactive phenotype in diabetes characterized by the induction of acute-phase response proteins and other inflammation-related genes. The concomitant upregulation of IL-1beta in the retina of diabetic rats points to this cytokine as a possible mediator of the acute-phase response mounted by Muller cells in diabetes.

The Relative Burden of Dry Eye in Patients' Lives: Comparisons to a U.S. Normative Sample

Abstract: PURPOSE. To assess the relative burden of dry eye in daily life by comparing Short Form-36 (SF-36) responses from individuals with and without dry eye against U.S. norms. METHODS. Assessment of 210 people, 130 with non-Sjogren's keratoconjunctivitis sicca (non-SS KCS), 32 with Sjogren's Syndrome (SS), and 48 control subjects. The study population data and published normative SF-36 data were compared. Dry eye severity was assessed by recruited severity (control, non-SS KCS, SS), patient self-report (none, very mild/mild, moderate, severe/extremely severe), and clinician-report (none, mild, moderate, severe). Age- and gender-matched norms were compared with all defined severity groups. RESULTS. Compared with the norms, control subjects scored higher on all SF-36 scales. Effect size (ES) ranged from 0.15 to 0.52. Non-SS KCS patients had lower Role-Physical (ES = -0.07), Bodily Pain (ES = -0.08), and Vitality (ES = -0.11) scores, indicating more dry eye impact on those areas versus the norm. All SF-36 scale scores except Mental Health (ES = 0.12) were lower in the SS group than the adjusted norm (ES range: -0.16 to -0.99). Regardless of severity classification, mild patients consistently had lower Role-Physical and Bodily Pain scores than the norm, suggesting impact on daily roles (ES < 0.2). Patients with moderately severe disease also experienced less vitality and poorer general health. The group with severe disease scored lower than the norm across all domains (ES range: -0.14 to -0.91) except Role-Emotional (ES = 0.13) and Mental Health (ES = 0.23). CONCLUSIONS. These results indicate dry eye's negative impact on everyday life, particularly in daily activities. Further research using disease-specific measures to examine dry eye's impact is underway.

Intravitreal injection of corticosteroid attenuates leukostasis and vascular leakage in experimental diabetic retina.

Abstract: PURPOSE Recently, intravitreal injection of corticosteroids has been in wide use as a treatment for diabetic macular edema, and the outcomes have been favorable. However, the exact mechanism remains unclear. The hypothesis for the current study was that intravitreal corticosteroids may improve diabetic retinal edema by amelioration of blood-retinal barrier (BRB) breakdown, by inhibiting leukocyte stasis (leukostasis). METHODS Diabetes was induced in 6-week-old male Long-Evans rats by intraperitoneal injection of streptozotocin (75 mg/kg). Three weeks after induction of diabetes, intravitreal injection of dexamethasone (40 microg/10 microL) was performed. At 2 days after intravitreal injection, accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and BRB breakdown was evaluated by measurement of retinal vascular permeability. The mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1 in the retina were also studied. RESULTS The number of leukocytes accumulated in the retina, once increased in the diabetic group, was decreased by 31.6% (P = 0.0001) after dexamethasone injection. The level of BRB breakdown, also elevated in the diabetic group, was suppressed by 61.1% (P = 0.0046) after dexamethasone injection. The level of ICAM-1 mRNA expression and its protein, upregulated in the diabetic group, were downregulated by dexamethasone treatment by 70.0% (P < 0.0001) and 56.4% (P = 0.0003). CONCLUSIONS Intravitreal injection of corticosteroids improves diabetic retinal edema through inhibiting leukocyte recruitment in the diabetic retina.

The Primordial, Blue Cone Color System of the Mouse Retina

Abstract: Humans and old world primates have trichromatic color vision based on three spectral types of cone [long-wavelength (L-), middle-wavelength (M-), and short-wavelength (S-) cones]. All other placental mammals are dichromats, and their color vision depends on the comparison of L- and S-cone signals; however, their cone-selective retinal circuitry is still unknown. Here, we identified the S-cone-selective (blue cone) bipolar cells of the mouse retina. They were labeled in a transgenic mouse expressing Clomeleon, a chloride-sensitive fluorescent protein, under the control of the thy1 promoter. Blue-cone bipolar cells comprise only 1-2% of the bipolar cell population, and their dendrites selectively contact S-opsin-expressing cones. In the dorsal half of the mouse retina, only 3-5% of the cones express S-opsin, and they are all contacted by blue-cone bipolar cells, whereas all L-opsin-expressing cones (∼95%) are avoided. In the ventral mouse retina, the great majority of cones express both S- and L-opsin. They are not contacted by blue-cone bipolar cells. A minority of ventral cones express S-opsin only, and they are selectively contacted by blue-cone bipolar cells. We suggest that these are genuine S-cones. In contrast to the other cones, their pedicles contain only low amounts of cone arrestin. The blue-cone bipolar cells of the mouse retina and their cone selectivity are closely similar to primate blue-cone bipolars, and we suggest that they both represent the phylogenetically ancient color system of the mammalian retina.

Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity.

Abstract: Purpose To determine the effects of solute molecular weight and lipophilicity on the permeability of a retinal pigment epithelium (RPE)-choroid preparation. Methods Fresh RPE-choroid specimens from bovine eyes were placed in diffusion chambers for permeability experiments with carboxyfluorescein, fluorescein isothiocyanate (FITC)-labeled dextrans with molecular masses from 4 to 80 kDa, and beta-blockers exhibiting a wide range of lipophilicity (atenolol, nadolol, pindolol, timolol, metoprolol, and betaxolol). Permeability experiments were performed both in the choroid-to-retina (inward) direction and in the retina-to-choroid (outward) direction. Carboxyfluorescein and FITC-dextrans were determined by fluorometry, and beta-blockers by HPLC. The transepithelial electrical resistance and potential difference were monitored during the experiments. Results Permeability of the fluorescent FITC-dextran probes through RPE-choroid decreased significantly with the increasing size of the probe. RPE-choroid was 35 times more permeable to carboxyfluorescein (376 Da) than to FITC-dextran 80 kDa. The permeabilities of lipophilic beta-blockers were up to 8 and 20 times higher than that of hydrophilic atenolol and carboxyfluorescein, respectively. The lag time of solute flux across the RPE-choroid increased with the molecular weight and lipophilicity. Compared with published data on isolated sclera, bovine RPE-choroid was 10 to 100 times less permeable to hydrophilic compounds and macromolecules. The permeability of lipophilic molecules in RPE-choroid was in the same range as in the sclera. Conclusions RPE is a major barrier and may be the rate-limiting factor in the retinal delivery of hydrophilic drugs and macromolecules through the transscleral route. For lipophilic molecules, RPE-choroid, and sclera are approximately equal barriers.