PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (899)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
more »
Document Types
1.  Stimulus-Evoked Intrinsic Optical Signals in the Retina: Spatial and Temporal Characteristics 
Purpose
To characterize the properties of stimulus-evoked retinal intrinsic signals and determine the underlying origins.
Methods
Seven adult cats were anesthetized and paralyzed to maximize imaging stability. The retina was stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera (Topcon, Tokyo, Japan). The LCD presented patterned visual stimuli while the retina was illuminated with near infrared (NIR) light. The peristimulus changes in the NIR reflectance of the retina were recorded with a digital camera.
Results
Two stimulus-evoked reflectance signals in the NIR were observed: a positive signal, corresponding to a relative increase in reflectance, and a negative signal, corresponding to a relative decrease in reflectance. When presented with a positive-contrast stimulus, the negative reflectance signals showed a tight spatial coupling with the stimulated region of retina, whereas the positive signals arose in an adjacent region of the retina. Signals remained spatially confined to the stimulated region even when stimuli of much longer duration were used. In addition, the positive and negative signal polarities reversed when the stimulus contrast was inverted. Both signals showed a rise time on the order of seconds, similar to those observed in the mammalian neocortex. The spectral dependency of the signals on illumination was similar to the absorbance spectra of hemoglobin and the oximetric relationship.
Conclusions
The findings characterize the basic properties of stimulus-evoked intrinsic signals of the retina. These signals were generally similar to the more extensively studied cortical signals. Collectively, the data suggest a hemodynamic component to the intrinsic optical signals of the retina.
doi:10.1167/iovs.08-3290
PMCID: PMC5201213  PMID: 19420337
2.  Stimulus-Evoked Intrinsic Optical Signals in the Retina: Pharmacologic Dissection Reveals Outer Retinal Origins 
Purpose
To elucidate the anatomic origins of stimulus-evoked intrinsic optical signals in the mammalian retina by using selective pharmacologic blockade of specific retinal layers.
Methods
Four adult cats were used to investigate the stimulus-evoked intrinsic signals. The retinas were visually stimulated with a liquid crystal display (LCD) integrated into a modified fundus camera. The evoked signals in the near infrared (NIR) were recorded with a digital camera to image the changes in the optical reflectance of the retinas. Variants of the electroretinogram (pattern ERG and long-pulse ERG) were also recorded as additional measures of retinal function. Specific retinal layers were inactivated via intravitreal injections of the voltage-gated sodium channel blocker, tetrodotoxin (TTX), the metabotropic glutamate receptor (mGluR6) agonist, 2-amino-4-phosphonobutyric acid (APB), and/or the ionotropic glutamate receptor antagonist cis-2,3 piperidinedicarboxylic acid (PDA). The stimulus-evoked intrinsic signals were imaged before and after drug injection.
Results
ERG recordings and tests of the consensual pupillary response confirmed the effectiveness of each drug. Yet despite the pharmacologic blockade of the inner retina (TTX) and postreceptoral retinal circuitry (APB and PDA), the stimulus-evoked intrinsic signals remained essentially unaltered from preinjection conditions. Similarly, the time course of the signal did not appreciably shift in time or shape.
Conclusions
The findings demonstrate that stimulus-evoked intrinsic signals persist after injection of APB, PDA, and TTX, drugs that work to suppress inner and postreceptoral retinal circuitry. The persistence of the intrinsic signals after administration of these drugs indicates that the dominant intrinsic signals are likely to arise from the outer retina.
doi:10.1167/iovs.08-3291
PMCID: PMC5201214  PMID: 19420331
3.  Timing of Surgery for Infantile Esotropia in Humans: Effects on Cortical Motion Visual Evoked Responses 
Purpose
Infantile esotropia is associated with maldevelopment of cortical visual motion processing, manifested as directional asymmetry of motion visual evoked potentials (mVEPs). The purpose of this study was to determine whether early surgery at or before age 11 months could promote the development of cortical visual motion processing in human infants, compared with standard surgery at age 11 to 18 months.
Methods
Sixteen children with a constant, infantile esotropia ≥30 prism diopters and onset before age 6 months were recruited prospectively. Eight of them underwent early surgery at ≤11 months of age, and eight underwent standard surgery at 11 to 18 months of age. Seven age-matched normal subjects served as the control. At 2 to 2.5 years of age, mVEPs were measured during monocular viewing of a grating that shifted between two positions with a lateral displacement of 90° at 10 Hz. Nasotemporal mVEP asymmetry was assessed by an amplitude asymmetry index and by the presence of a significant interocular phase difference.
Results
The mean asymmetry index and interocular phase difference in the early surgery group were comparable to that in age-matched control subjects, and they were significantly lower than those in the standard surgery group.
Conclusions
Early surgery for infantile esotropia promotes the development of cortical visual motion processing, whereas standard surgery is associated with abnormal mVEPs. The results provide additional evidence that early strabismus repair is beneficial for cortical development in human infants.
doi:10.1167/iovs.08-1836
PMCID: PMC5148621  PMID: 18441299 CAMSID: cams710
4.  Effects of Anisometropic Amblyopia on Visuomotor Behavior, I: Saccadic Eye Movements 
Purpose
Impairment of spatiotemporal visual processing is the hallmark of amblyopia, but its effects on eye movements during visuomotor tasks have rarely been studied. Here the authors investigate how visual deficits in anisometropic amblyopia affect saccadic eye movements.
Methods
Thirteen patients with anisometropic amblyopia and 13 control subjects participated. Participants executed saccades and manual reaching movements to a target presented randomly 5° or 10° to the left or right of fixation in three viewing conditions: binocular, amblyopic, and fellow eye viewing. Latency, amplitude, and peak velocity of primary and corrective saccades were measured.
Results
Initiation of primary saccades was delayed and more variable when patients viewed monocularly with their amblyopic eye. During binocular viewing, saccadic latency exhibited increased variability and no binocular advantage in patients (i.e., mean latency was similar to that during fellow eye viewing). Mean amplitude and peak velocity of primary saccades were comparable between patients and control subjects; however, patients exhibited greater variability in saccade amplitude. The frequency of corrective saccades was greater when patients viewed with their fellow eye than it was with binocular or amblyopic eye viewing. Latency, amplitude, and peak velocity of corrective saccades in patients were normal in all viewing conditions.
Conclusions
Saccades had longer latency and decreased precision in amblyopia. Once saccades were initiated, however, the dynamics of saccades were not altered. These findings suggest that amblyopia is associated with slower visual processing in the afferent (sensory) pathway rather than a deficit in the efferent (motor) pathway of the saccadic system.
doi:10.1167/iovs.10-5882
PMCID: PMC5142839  PMID: 20671288 CAMSID: cams1734
5.  Effects of Anisometropic Amblyopia on Visuomotor Behavior, Part 2: Visually Guided Reaching 
Purpose
The effects of impaired spatiotemporal vision in amblyopia on visuomotor skills have rarely been explored in detail. The goal of this study was to examine the influences of amblyopia on visually guided reaching.
Methods
Fourteen patients with anisometropic amblyopia and 14 control subjects were recruited. Participants executed reach-to-touch movements toward targets presented randomly 5° or 10° to the left or right of central fixation in three viewing conditions: binocular, monocular amblyopic eye, and monocular fellow eye viewing (left and right monocular viewing for control subjects). Visual feedback of the target was removed on 50% of the trials at the initiation of reaching.
Results
Reaching accuracy was comparable between patients and control subjects during all three viewing conditions. Patients’ reaching responses were slightly less precise during amblyopic eye viewing, but their precision was normal during binocular or fellow eye viewing. Reaching reaction time was not affected by amblyopia. The duration of the acceleration phase was longer in patients than in control subjects under all viewing conditions, whereas the duration of the deceleration phase was unaffected. Peak acceleration and peak velocity were also reduced in patients.
Conclusions
Amblyopia affects both the programming and the execution of visually guided reaching. The increased duration of the acceleration phase, as well as the reduced peak acceleration and peak velocity, might reflect a strategy or adaptation of feedforward/feedback control of the visuomotor system to compensate for degraded spatiotemporal vision in amblyopia, allowing patients to optimize their reaching performance.
doi:10.1167/iovs.10-6092
PMCID: PMC5115912  PMID: 21051723 CAMSID: cams1735
6.  Adaptive Neural Mechanism for Listing’s Law Revealed in Patients with Skew Deviation Caused by Brainstem or Cerebellar Lesion 
Purpose
Skew deviation is a vertical strabismus caused by damage to the otolithic–ocular reflex pathway and is associated with abnormal ocular torsion. This study was conducted to determine whether patients with skew deviation show the normal pattern of three-dimensional eye control called Listing’s law, which specifies the eye’s torsional angle as a function of its horizontal and vertical position.
Methods
Ten patients with skew deviation caused by brain stem or cerebellar lesions and nine normal control subjects were studied. Patients with diplopia and neurologic symptoms less than 1 month in duration were designated as acute (n = 4) and those with longer duration were classified as chronic (n = 10). Serial recordings were made in the four patients with acute skew deviation. With the head immobile, subjects made saccades to a target that moved between straight ahead and eight eccentric positions, while wearing search coils. At each target position, fixation was maintained for 3 seconds before the next saccade. From the eye position data, the plane of best fit, referred to as Listing’s plane, was fitted. Violations of Listing’s law were quantified by computing the “thickness” of this plane, defined as the SD of the distances to the plane from the data points.
Results
Both the hypertropic and hypotropic eyes in patients with acute skew deviation violated Listing’s and Donders’ laws—that is, the eyes did not show one consistent angle of torsion in any given gaze direction, but rather an abnormally wide range of torsional angles. In contrast, each eye in patients with chronic skew deviation obeyed the laws. However, in chronic skew deviation, Listing’s planes in both eyes had abnormal orientations.
Conclusions
Patients with acute skew deviation violated Listing’s law, whereas those with chronic skew deviation obeyed it, indicating that despite brain lesions, neural adaptation can restore Listing’s law so that the neural linkage between horizontal, vertical, and torsional eye position remains intact. Violation of Listing’s and Donders’ laws during fixation arises primarily from torsional drifts, indicating that patients with acute skew deviation have unstable torsional gaze holding that is independent of their horizontal–vertical eye positions.
doi:10.1167/iovs.07-0292
PMCID: PMC5104537  PMID: 18172094 CAMSID: cams723
7.  The Linear Vestibulo-Ocular Reflex in Patients with Skew Deviation 
Purpose
The linear vestibulo-ocular reflex (LVOR) is mediated primarily by the otolith organs in the inner ear. Skew deviation is a vertical strabismus believed to be caused by imbalance of otolithic projections to ocular motor neurons (disynaptically through the medial longitudinal fasciculus in the brain stem or polysynaptically through the cerebellum). The authors postulated that if skew deviation is indeed caused by damage to these projections, patients with skew deviation would show abnormal LVOR responses.
Methods
Six patients with skew deviation caused by brain stem or cerebellar lesions and 10 healthy subjects were recruited. All subjects underwent brief, sudden, interaural translations of the head (head heaves) using a head-sled device at an average peak acceleration of 0.42g (range, 0.1–1.1g) while continuously viewing an earth-fixed target monocularly at 15 and 20 cm. LVOR sensitivity (peak rotational eye velocity to peak linear head velocity) and velocity gain (peak actual-to-ideal rotational eye velocities) were calculated for the responses within the first 100 ms after onset of head movements.
Results
LVOR sensitivities and velocity gains in patients were decreased by 56% to 62% in both eyes compared with healthy subjects. This binocular reduction in LVOR responses was asymmetric—the magnitude of reduction differed between eyes by 37% to 143% for sensitivities and by 36% to 94% for velocity gains. There were no differences in response between right and left heaves.
Conclusions
The binocular, asymmetric reduction in LVOR sensitivity and velocity gain provides support that imbalance in the otolith-ocular pathway is a mechanism of skew deviation.
doi:10.1167/iovs.08-2254
PMCID: PMC5104545  PMID: 18775861 CAMSID: cams712
8.  Binocular Rivalry Measured 2 Hours After Occlusion Therapy Predicts the Recovery Rate of the Amblyopic Eye in Anisometropic Children 
Purpose
Recent studies on adults have shown that short-term monocular deprivation boosts the deprived eye signal in binocular rivalry, reflecting homeostatic plasticity. Here we investigate whether homeostatic plasticity is present also during occlusion therapy for moderate amblyopia.
Methods
Binocular rivalry and visual acuity (using Snellen charts for children) were measured in 10 children (mean age 6.2 ± 1 years) with moderate anisometropic amblyopia before the beginning of treatment and at four intervals during occlusion therapy (2 hours, 1, 2, and 5 months). Visual stimuli were orthogonal gratings presented dichoptically through ferromagnetic goggles and children reported verbally visual rivalrous perception. Bangerter filters were applied on the spectacle lens over the best eye for occlusion therapy.
Results
Two hours of occlusion therapy increased the nonamblyopic eye predominance over the amblyopic eye compared with pretreatment measurements, consistent with the results in adults. The boost of the nonamblyopic eye was still present after 1 month of treatment, steadily decreasing afterward to reach pretreatment levels after 2 months of continuous occlusion. Across subjects, the increase in nonamblyopic eye predominance observed after 2 hours of occlusion correlated (rho = −0.65, P = 0.04) with the visual acuity improvement of the amblyopic eye measured after 2 months of treatment.
Conclusions
Homeostatic plasticity operates during occlusion therapy for moderate amblyopia and the increase in nonamblyopic eye dominance observed at the beginning of treatment correlates with the amblyopic eye recovery rate. These results suggest that binocular rivalry might be used to monitor visual cortical plasticity during occlusion therapy, although further investigations on larger clinical populations are needed to validate the predictive power of the technique.
doi:10.1167/iovs.15-18419
PMCID: PMC4909145  PMID: 27046118
homeostatic plasticity; amblyopia; occlusion therapy; binocular rivalry; psychophysics
9.  A novel RPE65 hypomorph expands the clinical phenotype of RPE65 mutations. A comprehensive clinical and biochemical functional study 
Purpose
Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. We correlate the functional consequences of the novel P25L RPE65 mutation with its early childhood phenotype and compare it with other pathogenic missense mutations.
Methods
In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and 2-color-threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids.
Results
A very mild phenotype was detected in a now 7-y old boy homozygous for the P25L mutation in RPE65. Though abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5-y and 20/30 at age 7-y. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated answers under scotopic conditions, light-adapted cone answers were about 1.5 log units below normal. High resolution spectral domain OCT revealed morphological changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wildtype RPE65-transfected cells, while RPE65/L22P-transfected cells had 13.5%.
Conclusions
The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to < 10% of wildtype RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. We conclude that functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy.
doi:10.1167/iovs.07-1671
PMCID: PMC5015590  PMID: 18599565
10.  Morphological Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid-Stimulated Human Corneal Fibroblasts 
Purpose
To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures.
Methods
Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of Vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to five weeks. Constructs were assessed via light (phase contrast and differential interference contrast) and transmission (standard and quick freeze/deep etch) microscopy.
Results
Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) which often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct while quick-freeze deep etch micrographs showed the details of the matrix interaction with fibroblasts via arrays of membrane surface structures.
Conclusions
Human keratocytes, cultured in a stable Vitamin C derivative, are capable of assembling extracellular matrix which comprise parallel arrays of ECM fibrils. The resulting constructs, which are highly cellular, exhibit morphology similar to the developing mammalian stroma where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggest a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes which govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue-engineering a biomimetic stroma.
doi:10.1167/iovs.06-1216
PMCID: PMC4961093  PMID: 17724187
11.  Vascular Precursors in Developing Human Retina 
Purpose
Prior investigation has demonstrated that angioblasts are present in the inner retinas of human embryos and fetuses and that they differentiate and organize to form the primordial retinal vasculature. The purpose of this study was to characterize these angioblasts further and examine ligands that might control their migration and differentiation.
Methods
Immunohistochemistry was used to localize stroma-derived factor-1 (SDF-1), its receptor CXCR4, stem cell factor (SCF), and its receptor c-Kit on sections obtained from human eyes at from 6 to 23 weeks’ gestation (WG). Coexpression of CD39 (marker for retinal angioblasts and endothelial cells) and CXCR4 or c-Kit was investigated by confocal microscopy.
Results
SDF-1 was prominent in inner retina with the greatest reaction product near the internal limiting membrane (ILM). SCF immunoreactivity was also confined to the inner retina and increased significantly between 7 and 12 WG. The level of both ligands declined by 22 WG. A layer of CXCR4+ and c-Kit+ precursors, some of which coexpressed CD39, existed in the inner retina from 7 to 12 WG. With migration, c-Kit was downregulated, whereas CD39+ cells continued to express CXCR4 as they formed cords. With canalization, CXCR4 expression was downregulated.
Conclusions
Embryonic human retina has a pool of precursors (CXCR4+ and c-Kit+) that enlarged centrifugally during fetal development. From this pool emerges angioblasts, which migrate anteriorly into the nerve fiber layer where SDF-1 and SCF levels are highest. c-Kit expression declines with apparent migration, and CXCR4 expression declines with canalization of new vessels. Both SCF and SDF-1 are associated with the differentiation of retinal precursors into angioblasts and their migration to sites of vessel assembly.
doi:10.1167/iovs.07-0632
PMCID: PMC4943084  PMID: 18436851
12.  Structural and functional maturation of the fetal human choriocapillaris 
Purpose
The purpose of this study was to examine the structural and functional maturation of the choriocapillaris (CC). We sought to determine when fenestrations formed, pericytes invest the capillaries and endothelial cells became functional.
Methods
Immunohistochemistry was performed on cryopreserved sections of embryonic/fetal human eyes from 7 to 22 weeks gestation (WG) using antibodies against PAL-E, PV-1 (fenestrations), carbonic anhydrase IV (CA IV), eNOS, and alpha smooth muscle actin (αSMA) and NG2 (two pericyte markers) and endothelial cell (EC) markers (CD34, CD31). Alkaline phosphatase (APase) enzymatic activity was demonstrated by enzyme histochemistry. Transmission electron microscopy (TEM) was performed on 11, 14, 16 and 22 WG eyes. Adult human eyes were used as positive controls.
Results
All EC markers were present in CC by 7 WG. PAL-E, CA IV and eNOS immunoreactivities and APase activity were present in CC by 7–9 WG. TEM analysis demonstrated how structurally immature this vasculature was even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. The few fenestrations that were observed were often present within the luminal space in the filopodia. Contiguous fenestrations and significant PV-1 were not observed until 21–22 WG. αSMA was prominent at 22 WG and the maturation of pericytes was confirmed by TEM.
Conclusions
It appears that EC and their precursors have several mature functional characteristics well before they are structurally mature. Although EC make tight junctions early in development, contiguous fenestrations and mature pericytes occur much later in development.
doi:10.1167/iovs.08-2614
PMCID: PMC4941237  PMID: 19264887
choriocapillaris; fenestrations; fetal; immunohistochemistry; pericytes; ultrastructure
13.  Posterior Corneal Aberrations and Their Compensation Effects on Anterior Corneal Aberrations in Keratoconic Eyes 
Purpose
To characterize posterior corneal aberrations in keratoconic (KC) eyes and investigate compensatory effects between anterior and posterior corneal surfaces.
Methods
The corneal topography of 113 eyes (37 advanced KC, 31 moderate KC, 14 mild KC, and 31 normal eyes) was used to compute the corneal aberrations. The central 6-mm diameter of both anterior and posterior corneal topographies was decomposed into Zernike polynomials. The magnitude and the orientation of each posterior corneal aberration were calculated by vector analysis. The compensation effects between anterior and posterior corneal aberrations were also assessed quantitatively with a linear regression model.
Results
The average higher order RMS wavefront errors for the posterior corneas were 1.04, 0.54, 0.24, and 0.19 µm in the advanced, moderate, and mild KC and normal eyes, respectively. In the advanced KC eyes, posterior corneal coma was oriented in the superior–nasal direction with a mean orientation angle of 75° ± 19° OD and 78° ± 20° OS. On average, 22%, 24%, and 14% of the anterior corneal coma were compensated by the posterior cornea in the advanced, moderate, and mild KC eyes, respectively. However, no significant higher order aberration (HOA) compensation effects were found in normal corneas.
Conclusions
Significantly larger amounts of posterior corneal aberrations and stronger compensation effects were observed in KC eyes than in normal eyes. The uncorrected posterior corneal aberration in KC eyes was substantial and degraded retinal image quality. This may explain the relatively poor visual acuity obtained in eyes with rigid gas permeable (RGP) lenses, which correct only anterior corneal aberrations.
doi:10.1167/iovs.08-1874
PMCID: PMC4870836  PMID: 18641282
14.  Adaptive Optics Scanning Laser Ophthalmoscopy Images in a Family with the Mitochondrial DNA T8993C Mutation 
Purpose
This study was designed to assess the effect of mitochondrial DNA (mtDNA) mutation T8993C on cone structure in a family expressing neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome.
Methods
Five family members were studied, using clinical examination, nerve conduction studies, perimetry, optical coherence tomography (OCT) measures of central retinal thickness, and electroretinography. High-resolution images of cone structure using adaptive optics scanning laser ophthalmoscopy (AOSLO) were obtained in four subjects with stable fixation. Cone spacing was compared to 18 age-similar normal subjects and converted to z-scores at each location where unambiguous cones were identified. Tissue levels of T8993C mutant heteroplasmy in blood and hair follicles were quantified using real-time allele-refractory mutations system (ARMS) quantitative polymerase chain reaction (qPCR).
Results
Subjects expressing the T8993C mutation showed varying levels of disease severity. The subject with the lowest mutant load (42%–54%) showed no neurologic or retinal abnormalities. The remaining four subjects with over 77% mutant load all expressed severe neurologic and/or retinal abnormalities. AOSLO images revealed three patterns of cone spacing: pattern 1, normal; pattern 2, increased cone spacing within a contiguous cone mosaic; and pattern 3, patchy cone loss with increased cone spacing. Visual function was most severely affected in pattern 3.
Conclusions
High levels of T8993C mutant load were associated with severe neurologic or visual dysfunction, while lower levels caused no detectable abnormalities. Visual function was better in patients with a contiguous and regular cone mosaic. Patients expressing high levels of the mtDNA T8993C mutation show abnormal cone structure, suggesting normal mitochondrial DNA is necessary for normal waveguiding by cones.
doi:10.1167/iovs.08-2029
PMCID: PMC4836613  PMID: 18997096
15.  Relationship between RPE and choriocapillaris in age-related macular degeneration 
Purpose
The purpose of this study was to examine the relationships between choriocapillaris (CC) and retinal pigment epithelial (RPE) changes in age-related macular degeneration (AMD). Morphological changes in the RPE/choriocapillaris complex were quantified in dry and wet forms of AMD and the results compared to with aged control eyes without maculopathy.
Methods
Postmortem choroids from 3 aged control subjects, 5 geographic atrophy (GA) subjects and 3 wet AMD subjects were analyzed using a semi-quantitative computer-assisted morphometric technique developed to measure percent RPE and CC areas in choroidal whole mounts incubated for alkaline phosphatase activity. The tissues were subsequently embedded in methacrylate and sectioned to examine structural changes.
Results
There was a linear relationship between the loss of RPE and CC in GA. A 50% reduction in vascular area was found in regions of complete RPE atrophy. Extreme constriction of remaining viable capillaries was found in areas devoid of RPE. Adjacent to active choroidal neovascularization (CNV) in wet AMD, CC dropout was evident in the absence of RPE atrophy resulting in a 50% decrease in vascular area. Lumenal diameters of the remaining capillaries in wet AMD eyes were similar to controls.
Conclusions
The primary insult in GA appears to be at the level of the RPE and there is an intimate relationship between RPE atrophy and secondary CC degeneration. CC degeneration occurs in the presence of viable RPE in wet AMD. The RPE in regions of vascular dropout are presumably hypoxic, which may result in an increase in VEGF production by the RPE and stimulation of CNV.
doi:10.1167/iovs.09-3639
PMCID: PMC4829357  PMID: 19357355
age-related macular degeneration; geographic atrophy; choriocapillaris; retinal pigment epithelium; choroidal neovascularization
16.  Ultrastructural and molecular biologic comparison of classical proprioceptors and palisade endings in sheep extraocular muscles 
Purpose
To analyze and compare the structural and molecular features of classical proprioceptors like muscle spindles and Golgi tendon organs (GTOs) and putative proprioceptors (palisade endings) in sheep extraocular muscle (EOMs).
Methods
The EOMs of four sheep were analyzed. Frozen sections or whole mount preparations of the samples were immunohistochemically labeled and analyzed by confocal laser scanning microscopy. Triple labeling with different combinations of antibodies against neurofilament, synaptophysin and choline acetyltransferase (ChAT) as well as α-bungarotoxin and phalloidin was performed. Microscopic anatomy of the nerve end organs was analyzed by transmission electron microscopy.
Results
The microscopic anatomy demonstrated that muscle spindles and GTOs had a perineural capsule and palisade endings a connective tissue capsule. Sensory nerve terminals in muscle spindles and GTOs contained only few vesicles whereas palisade nerve terminals were full of clear vesicles. Likewise, motor terminals in the muscle spindles’ polar regions were full of clear vesicles. Immunohistochemistry showed that sensory nerve fibers as well as their sensory nerve terminals in muscle spindles and GTOs were ChAT-negative. Palisade endings were supplied by ChAT-positive nerve fibers and the palisade complexes including palisade nerve terminals were also ChAT-immunoreactive. Motor terminals in muscle spindles were ChAT and α-bungarotoxin -positive.
Conclusions
The present study demonstrated in sheep EOMs that palisade endings are innervated by cholinergic axons exhibiting characteristics typical for motoneurons whereas muscle spindles (except the polar regions) and GTOs are supplied by non-cholinergic axons. These results question whether palisade endings are candidates for proprioceptors in EOMs.
doi:10.1167/iovs.09-3902
PMCID: PMC4811329  PMID: 19553627
sheep; eye movement; proprioception; palisade endings; Golgi tendon organs; muscle spindles; choline acetyltransferase
17.  Nuclear Ferritin: A Ferritoid-Ferritin Complex in Corneal Epithelial Cells 
Purpose
Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes.
Methods
For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay.
Results
The CE nuclear ferritin complex has characteristics that differentiate it from a “typical” cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA.
Conclusions
Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.
doi:10.1167/iovs.08-3170
PMCID: PMC4793724  PMID: 19255152
18.  Reduction of the Available Area for Aqueous Humor Outflow and Increase in Meshwork Herniations into Collector Channels Following Acute IOP Elevation in Bovine Eyes 
Purpose
To understand how hydrodynamic and morphologic changes in the aqueous humor outflow pathway contribute to decreased aqueous humor outflow facility after acute elevation of intraocular pressure (IOP) in bovine eyes.
Methods
Enucleated bovine eyes were perfused at 1 of 4 different pressures (7, 15, 30, 45 mm Hg) while outflow facility was continuously recorded. Dulbecco PBS + 5.5 mM glucose containing fluorescent microspheres (0.5 μm, 0.002% vol/vol) was perfused to outline aqueous outflow patterns, followed by perfusion-fixation. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in radial and frontal sections. Percentage effective filtration length (PEFL; IW length exhibiting tracer labeling/total length of IW) was measured. Herniations of IW into collector channel (CC) ostia were examined and graded for each eye by light microscopy.
Results
Increasing IOP from 7 to 45 mm Hg coincided with a twofold decrease in outflow facility (P < 0.0001), a 33% to 57% decrease in PEFL with tracer confined more to the vicinity of CC ostia, progressive collapse of the AP, and increasing percentage of CC ostia exhibiting herniations (from 15.6% ± 6.5% at 7 mm Hg to 95% ± 2.3% at 30 mm Hg [P < 10−4], reaching 100% at 45 mm Hg).
Conclusions
Decreasing outflow facility during acute IOP elevation coincides with a reduction in available area for aqueous humor outflow and the confinement of outflow to the vicinity of CC ostia. These hydrodynamic changes are likely driven by morphologic changes associated with AP collapse and herniation of IW of AP into CC ostia.
doi:10.1167/iovs.08-1707
PMCID: PMC4788035  PMID: 18515571
19.  Hypoxia-Regulated Activity of PKCε in the Lens 
Purpose
To show that hypoxia is necessary to prevent opacification of the lens. Protein kinase C (PKC)-ε serves a role that is distinct from PKC-γ when both PKC isoforms are expressed in the lens. PKCε serves a very important role in hypoxic conditions, helping to prevent opacification of the lens.
Methods
Digital image analysis, confocal microscopy, dye transfer assay, coimmunoprecipitation, Western blot analysis, and enzyme activity assays were used, respectively, to study opacification of the lens, intercellular communications, cellular localization of connexin-43 (Cx43), and the interactions between PKCε, PKCγ, and Cx43 in the lens epithelial cells.
Results
Hypoxic conditions (1%–5% of oxygen) were very important in maintaining clarity of the lenses of wild-type (WT) mice. Normoxic conditions induced opacification of the WT lens. Lenses from the PKCε-knockout mice underwent rapid opacification, even in hypoxic conditions. Hypoxia did not induce apoptosis in the lens epithelial cells, judging by the absence of active caspase-3, and it did not change intercellular communication and did not affect the number and localization of junctional Cx43 plaques in the lens epithelial cell culture. Hypoxia activated PKCε, whereas phorbol ester (TPA), oxidation (H2O2), and insulin-like growth factor-1 (IGF-1) activated PKCγ and decreased the activity of PKCε. Hypoxia did not induce the phosphorylation of the Cx43.
Conclusions
Hypoxia-induced activation of PKCε is very important in surviving hypoxia and maintaining the clarity of the lens. However, PKCγ is utilized in the control of Cx43 gap junctions.
doi:10.1167/iovs.08-2599
PMCID: PMC4762488  PMID: 18997087
20.  Collision judgment when using an augmented-vision head-mounted display device 
Purpose
We have developed a device to provide an expanded visual field to patients with tunnel vision by superimposing minified edge images of the wide scene, in which objects appear closer to the heading direction than they really are. We conducted experiments in a virtual environment to determine if users would overestimate collision risks.
Methods
Given simulated scenes of walking or standing with intention to walk towards a given direction (intended walking) in a shopping mall corridor, participants (12 normally sighted and 7 with tunnel vision) reported whether they would collide with obstacles appearing at different offsets from variable walking paths (or intended directions), with and without the device. The collision envelope (CE), a personal space based on perceived collision judgments, and judgment uncertainty (variability of response) were measured. When the device was used, combinations of two image scales (5× minified and 1:1) and two image types (grayscale or edge images) were tested.
Results
Image type did not significantly alter collision judgment (p>0.7). Compared to the without-device baseline, minification did not significantly change the CE of normally sighted subjects for simulated walking (p=0.12), but increased CE by 30% for intended walking (p<0.001). Their uncertainty was not affected by minification (p>0.25). For the patients, neither CE nor uncertainty was affected by minification (p>0.13) in both walking conditions. Baseline CE and uncertainty were greater for patients than normally-sighted subjects in simulated walking (p=0.03), but the two groups were not significantly different in all other conditions.
Conclusion
Users did not substantially overestimate collision risk, as the 5× minified images had only limited impact on collision judgments either during walking or before starting to walk.
doi:10.1167/iovs.08-2916
PMCID: PMC4754790  PMID: 19458339
21.  Convergence and accommodation development is pre-programmed in premature infants 
Purpose
This study investigated whether vergence and accommodation development in pre-term infants is pre-programmed or is driven by experience.
Methods
32 healthy infants, born at mean 34 weeks gestation (range 31.2-36 weeks) were compared with 45 healthy full-term infants (mean 40.0 weeks) over a 6 month period, starting at 4-6 weeks post-natally. Simultaneous accommodation and convergence to a detailed target were measured using a Plusoptix PowerRefII infra-red photorefractor as a target moved between 0.33m and 2m. Stimulus/response gains and responses at 0.33m and 2m were compared by both corrected (gestational) age and chronological (post-natal) age.
Results
When compared by their corrected age, pre-term and full-term infants showed few significant differences in vergence and accommodation responses after 6-7 weeks of age. However, when compared by chronological age, pre-term infants’ responses were more variable, with significantly reduced vergence gains, reduced vergence response at 0.33m, reduced accommodation gain, and increased accommodation at 2m, compared to full-term infants between 8-13 weeks after birth.
Conclusions
When matched by corrected age, vergence and accommodation in pre-term infants show few differences from full-term infants’ responses. Maturation appears pre-programmed and is not advanced by visual experience. Longer periods of immature visual responses might leave pre-term infants more at risk of development of oculomotor deficits such as strabismus.
doi:10.1167/iovs.14-15358
PMCID: PMC4549904  PMID: 26275135
Convergence; Accommodation; Development; Prematurity; Strabismus
22.  Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals 
Purpose
To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs).
Methods
Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done.
Results
In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin.
Conclusions
Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.
doi:10.1167/iovs.15-18716
PMCID: PMC4826744  PMID: 26830369
oculomotor system; proprioception; frontal-eyed species; lateral-eyed species; palisade endings; convergence
23.  A Drug-Eluting Contact Lens 
Purpose
To formulate and characterize a drug-eluting contact lens designed to provide extended, controlled release of a drug.
Methods
Prototype contact lenses were created by coating PLGA (poly[lactic-co-glycolic acid]) films containing test compounds with pHEMA (poly[hydroxyethyl methacrylate]) by ultraviolet light polymerization. The films, containing encapsulated fluorescein or ciprofloxacin, were characterized by scanning electron microscopy. Release studies were conducted in phosphate-buffered saline at 37°C with continuous shaking. Ciprofloxacin eluted from the contact lens was studied in an antimicrobial assay to verify antimicrobial effectiveness.
Results
After a brief and minimal initial burst, the prototype contact lenses demonstrated controlled release of the molecules studied, with zero-order release kinetics under infinite sink conditions for over 4 weeks. The rate of drug release was controlled by changing either the ratio of drug to PLGA or the molecular mass of the PLGA used. Both the PLGA and the pHEMA affected release kinetics. Ciprofloxacin released from the contact lenses inhibited ciprofloxacin-sensitive Staphylococcus aureus at all time-points tested.
Conclusions
A prototype contact lens for sustained drug release consisting of a thin drug-PLGA film coated with pHEMA could be used as a platform for ocular drug delivery with widespread therapeutic applications.
doi:10.1167/iovs.08-2826
PMCID: PMC4657544  PMID: 19136709
24.  Downregulation of Reduced-Folate Transporter by Glucose in Cultured RPE Cells and in RPE of Diabetic Mice 
PURPOSE
The polarized distribution of reduced-folate transporter (RFT)-1 to the apical retinal pigment epithelial (RPE) membrane was demonstrated recently. Nitric oxide (NO) significantly decreases the activity of RFT-1 in cultured RPE cells. NO is elevated in diabetes, and therefore in the present study the alteration of RFT-1 activity in RPE under conditions of high glucose was investigated.
METHODS
Human ARPE-19 cells were incubated in media containing 5 mM glucose plus 40 mM mannitol (control) or 45 mM glucose for varying periods and the activity of RFT-1 was assessed by determining the uptake of [3H]-N5-methyltetrahy-drofolate (MTF). The levels of mRNA encoding RFT-1 were determined by RT-PCR and protein levels by Western blot analysis. The activity of RFT-1 and expression of mRNA encoding RFT-1 were analyzed also in RPE of streptozotocin-induced diabetic mice.
RESULTS
Exposure of RPE cells to 45 mM glucose for as short an incubation time as 6 hours resulted in a 35% decrease in MTF uptake. Kinetic analysis showed that the hyperglycemia-induced attenuation was associated with a decrease in the maximal velocity of the transporter with no significant change in the substrate affinity. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was significantly decreased in cells exposed to high glucose, and Western blot analysis showed a significant decrease in protein levels. The uptake of [3H]-MTF in RPE of diabetic mice was reduced by approximately 20%, compared with that in nondiabetic, age-matched control animals. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was decreased significantly in RPE of diabetic mice.
CONCLUSIONS
These findings demonstrate for the first time that hyperglycemic conditions reduce the expression and activity of RFT-1 and may have profound implications for the transport of folate by RPE in diabetes.
PMCID: PMC4639924  PMID: 11818404
25.  Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro 
PURPOSE
Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC.
METHODS
In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells.
RESULTS
Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA.
CONCLUSIONS
This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required.
doi:10.1167/iovs.14-15973
PMCID: PMC4506787  PMID: 26024089
Stem cell; cornea; quantum dot; cell tracking; cell transplantation; limbal epithelium

Results 1-25 (899)