Elevated M2 Macrophage Markers in Epiretinal Membranes With Ectopic Inner Foveal Layers.

Purpose: To analyze the differences in the vitreous cytokine profiles in epiretinal membrane eyes with and without an ectopic inner foveal layer (EIFL). Methods: Sixty eyes with epiretinal membrane (32 eyes without EIFL and 28 eyes with EIFL) were included. The vitreous samples were collected during surgery for epiretinal membrane. The cytokine levels of the vitreous were measured using a multiplex bead analysis. Results: The mean logMAR visual acuity was worse (0.42 vs. 0.37; P = 0.331) and the central foveal thickness was higher in the EIFL group (496.9 µm vs. 434.2 µm; P = 0.007) than they were in the group without EIFL. The mean EIFL thickness was 164.1 ± 67.7 µm in the EIFL group. On multiplex analysis of the vitreous cytokines, the levels of CD163 (21529 pg/dL vs. 10877 pg/dL; P = 0.002) and macrophage colony-stimulating factor (206 pg/dL vs. 159 pg/dL, P = 0.004) were significantly higher in the EIFL group than they were in the group without EIFL. Conclusions: Eyes with EIFL had increased vitreous levels of M2 macrophage markers. The activation of glial cell proliferation by M2 macrophages may contribute to EIFL formation.

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer.

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

Foveal anatomic associations with the secondary peak and the slope of the macular pigment spatial profile.

PURPOSE: To investigate the reproducibility of the macular pigment (MP) spatial profile by using heterochromatic flicker photometry (HFP) and to relate the MP spatial profile to foveal architecture. METHODS: Sixteen healthy subjects (nine had the typical exponential MP spatial profile [group 1]; seven had a secondary peak MP spatial profile [group 2]) were recruited. The MP spatial profile was measured on three separate occasions. Six radiance measurements were obtained at each locus (0.25 degrees , 0.5 degrees , 1 degrees , and 1.75 degrees eccentricity; reference point, 7 degrees ). Foveal architecture was assessed by optical coherence tomography (OCT). RESULTS: Subjects who had the typical decline profile, had this profile after averaging repeated measures (group 1). Subjects who had a secondary peak, displayed the secondary peak after repeated measures were averaged (group 2). Mean SD foveal width in group 1 was significantly narrower than mean SD foveal width in group 2 (1306 +/- 240 microm and 1915 +/- 161 microm, respectively; P < 0.01). This difference remained after adjustment for sex (P < 0.001). Foveal width was significantly related to mean foveal MP, with adjustment for sex (r = 0.588, P = 0.021). Foveal profile slope was significantly related to MP spatial profile slope, after removal of an outlier (r = 0.591, P = 0.020). CONCLUSIONS: HFP reproducibly measures MP spatial profile. Secondary peaks seen in the MP spatial profile cannot be attributed to measurement error and are associated with wider foveas. The slope of an individual's MP spatial profile is related to foveal slope, with a steeper MP distribution associated with a steeper foveal depression.

Macular pigment optical density and photophobia light threshold.

Light absorption by macular pigment may attenuate visual discomfort, or photophobia, for targets composed of short-wavelength light. Macular pigment optical density (MPOD) and photophobia light thresholds were measured psychophysically in 10 subjects. The energy necessary to induce photophobia for a short-wavelength target relative to a long-wavelength target was linearly related to MPOD, as well as estimates of peak MPOD and integrated macular pigment. In four subjects who consumed lutein supplements, increases in MPOD corresponded to increases in photophobia light thresholds. Light absorption by macular pigment appears to influence the amount of short-wavelength light necessary to elicit photophobia.

Chiasmal misrouting and foveal hypoplasia without albinism.

BACKGROUND/AIMS: To present the ophthalmological and electrophysiological characteristics of three darkly pigmented, female patients with misrouting and foveal hypoplasia. One of the patients had primary ciliary dyskinesia and situs inversus totalis (Kartagener syndrome). METHODS: Fundus photographs were taken and the angles at which the main temporal arterial branches leave the optic nerve head (ONH) were analysed. Optical coherence tomography (OCT) was performed through the presumed foveal region. Pattern onset visually evoked potentials (VEPs) (check sizes 60', 40/400 ms) were recorded and the chiasmal coefficient was calculated to detect misrouting. RESULTS: Fundus photography showed normally pigmented fundi with absence of the usual foveal hyperpigmentation, foveal avascular zone, and macular and foveal reflexes. On OCT no foveal pit was found. The VEP recordings showed the largest positive CI component over the right hemisphere for the left eye, and over the left hemisphere for the right eye, with the CI almost absent over the ipsilateral hemispheres. The differential derivations showed opposite polarity for the recordings of the two eyes. The chiasmal coefficients of all three patients were significantly indicative of misrouting (-0.99, -0.91, and -0.99, respectively). CONCLUSION: Based on the investigations in these patients the authors propose the hypothesis that foveal hypoplasia and misrouting exist as a distinct entity, and do not comprise the exclusive hallmark of albinism. The findings suggest that misrouting may exert a retrograde influence on foveal development.

Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis.

PURPOSE: Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina. METHODS: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm. RESULTS: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images. CONCLUSIONS: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.

Autofluorescence characteristics of normal foveas and reconstruction of foveal autofluorescence from limited data subsets.

PURPOSE: To develop mathematical and geometric models of the nonuniform autofluorescence (AF) patterns of foveas of normal subjects and to reconstruct these models from limited subsets of data. METHODS: Confocal scanning laser ophthalmoscope (cSLO) AF fundus images of normal maculae were obtained from both eyes of 10 middle-aged subjects. They were filtered and contrast enhanced, to obtain elliptical isobars of equal gray levels (GLs) and determine the isobars' resolutions, eccentricities, and angles of orientation. The original image data were fit with a mathematical model of elliptic quadratic polynomials in two equal zones: the center and the remaining annulus. RESULTS: The AF images segmented into nested concentric GL isobars with GLs that increased radially from the least-fluorescent center. The mean isobar resolution was 31 +/- 7 mum. The geometric eccentricity of the ellipses increased from 0.42 +/- 0.12 centrally to 0.52 +/- 0.14 peripherally (P = 0.0005), with mean axes of orientation peripherally 97.12 +/- 15.46 degrees . The model fits to the complete image data had mean absolute normalized errors ranging from 3.6% +/- 3.7% to 7.3% +/- 7.1%. The model fits to small subsets (1% to 2% of total image data) had mean absolute errors ranging from 3.7% +/- 3.8% to 7.3% +/- 7.2%. CONCLUSIONS: Normal AF fundus images show finely resolved, concentric, elliptical foveal patterns consistent with the anatomic distribution of fluorescent lipofuscin, light-attenuating macular pigment (MP), cone photopigment, and retinal pigment epithelial (RPE) pigment in the fovea. A two-zone, elliptic, quadratic polynomial model can accurately model foveal data. This model may be useful for image analysis and for automated segmentation of pathology.

Müller cells in the human foveal region.

PURPOSE: To investigate the distribution of Müller cells in the foveal region of the human retina. METHODS: After fixation with 4% glutaraldehyde, the percentages of the area of Müller cells were calculated at the macula, posterior pole, equator, and periphery by electron microscopy. After fixation with 4% paraformaldehyde, the silver enhancing technique was applied to show glutamine synthetase (GS) and L-glutamate/L-apartate transporter (GLAST). Furthermore, for the solubilized retinas at each region, Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect GS and GLAST in the extracts. RESULTS: The percentages of the area of Müller cells in the outer nuclear layer (ONL) were 12.3 +/- 2.7, 22.1 +/- 4.5, 23.6 +/- 1, and 26.7 +/- 4.5%, respectively. We confirmed less GS and GLAST immunoreactivity in the foveal region. ELISA and Western blot analysis revealed that the amounts of GS and GLAST in the foveal region were smaller than those in any other region. CONCLUSIONS: These results showed that the density of Müller cells is low in the foveal region.

Influence of lutein supplementation on macular pigment, assessed with two objective techniques.

PURPOSE: Macular pigment (MP) may protect against age-related macular degeneration. This study was conducted to determine the extent of changes in the macular pigment density as a consequence of oral supplementation with lutein. A second purpose was to compare two objective measurement techniques. METHODS: In the first technique, reflectance maps were made with a scanning laser ophthalmoscope. Digital subtraction of log reflectance maps and comparison between the foveal area and a 14 degrees temporal site provided MP density estimates. In the second technique, spectral fundus reflectance of the fovea was measured with a fundus reflectometer and analyzed with a detailed optical model, to arrive at MP density values. Eight subjects participated in this study. They took 10 mg lutein per day for 12 weeks. Plasma lutein concentration was measured at 4-week intervals. RESULTS: After 4 weeks, mean blood level of lutein had increased from 0.18 to 0.90 microM. It stayed at this level throughout the intake period and declined to 0.28 microM 4 weeks after termination. Measurement of the density of MP showed a within-subject variation of 10% with MP maps and 17% with spectral reflectance analysis. MP density showed a mean linear 4-week increase of 5.3% (P: < 0.001) and 4.1% (P: = 0. 022), respectively. CONCLUSIONS: Supplementation with lutein significantly increased the density of the MP. Analyzing reflectance maps with a scanning laser ophthalmoscope provided very reliable estimates of MP.

Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina.

PURPOSE: In addition to acting as an optical filter, macular (carotenoid) pigment has been hypothesized to function as an antioxidant in the human retina by inhibiting the peroxidation of long-chain polyunsaturated fatty acids. However, at its location of highest density in the inner (prereceptoral) layers of the foveal retina, a specific requirement for antioxidant protection would not be predicted. The purpose of this study was to determine whether lutein and zeaxanthin, the major carotenoids comprising the macular pigment, are present in rod outer segment (ROS) membranes where the concentration of long-chain polyunsaturated fatty acids, and susceptibility to oxidation, is highest. METHODS: Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes. RESULTS: ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations. CONCLUSIONS: The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration.

Maturational gradients in the retina of the ferret.

In the present set of studies, we have examined the site for the initiation of retinal maturation in the ferret. A variety of maturational features across the developing inner and outer retina were examined by using standard immunohistochemical, carbocyanine dye labelling, and Nissl-staining techniques, including 1) two indices of early differentiation of the first-born retinal ganglion cells, the presence of beta-tubulin and of neuron-specific enolase; 2) the receding distribution of chondroitin sulfate proteoglycans within the inner retina; 3) the distribution of the first ganglion cells to grow axons along the optic nerve; 4) the emergence of the inner plexiform layer; 5) the emergence of the outer plexiform layer and 6) the onset of synaptophysin immunoreactivity within it; 7) the differentiation of calbindin-immunoreactive horizontal cells; and 8) the cessation of proliferative activity at the ventricular surface. Although we were able to define distinct maturational gradients that are associated with many of these features of inner and outer retinal development (each considered in detail in this report), with dorsal retina maturing before ventral retina, and with peripheral retina maturing last, none showed a clear initiation in the region of the developing area centralis. Rather, maturation began in the peripapillary retina dorsal to the optic nerve head, which is consistent with previous studies on the topography of ganglion cell genesis in the ferret. These results make clear that the order of retinal maturation and the formation of the area centralis are not linked, at least not in the ferret.

Characterization of a human fovea cDNA library and regional differential gene expression in the human retina.

The primate fovea is the region of the retina responsible for acute vision. This region constitutes less than 5% of the total area of the retina and has not been intensely studied at the molecular level. As a first step in the molecular characterization of the fovea, we have constructed a primary human fovea cDNA library. Experiments confirm that our cDNA library reflects a nonbiased distribution of foveal expressed sequences. Single-pass sequencing was performed on 209 randomly isolated clones from this library. Analysis of the sequences generated reveals that the distributions of fovea clones with either human mitochondrial gene sequences or repetitive elements are different than those observed in cDNA libraries made from other tissues. A significant number of the fovea expressed sequence tags (ESTs) (88, 42.1%) represent novel human ESTs. This suggests that the library will be useful in identifying new human genes. Northern analysis of specific fovea ESTs defined in this study suggests that there are significant quantitative differences in gene expression that distinguish the fovea from the rest of the retina.

Isolation of differentially expressed human fovea genes: candidates for macular disease.

PURPOSE: In humans, the fovea is the region of the retina responsible for acute vision. Disorders affecting the fovea are responsible for the majority of cases of untreated blindness in the developed world, yet are poorly understood at the molecular level. Our goal is to identify genes that are preferentially expressed within the human fovea as compared to the midperipheral retina (differential fovea clones). MATERIALS AND METHODS: An unamplified fovea cDNA library was differentially screened with cDNA probes derived from either human fovea or midperipheral retina. Rounds of secondary screening and northern analysis were used to verify the expression pattern of a selective number of clones isolated. RESULTS: Forty-one differential fovea clones were isolated from a screening of 10,000 phage clones (clones). Of these clones, 31.5 % correspond to known sequences present in GenBank/EMBL and 70.7% represent novel human fovea expressed sequence tags (ESTs). Northern analysis of selected clones demonstrated that they represent genes expressed at higher levels in the human fovea than in the midperipheral retina. CONCLUSIONS: Genes that are more highly expressed in the fovea as opposed to the midperipheral retina are likely to represent essential genes for fovea function. Using our fovea cDNA library, we are able to isolate differential human fovea clones at an incidence of 41/10,000 clones screened. We demonstrate that there is a high level of differential gene expression within different regions of the human retina.

Presence and foveal enrichment of rod opsin in the "all cone" retina of the American chameleon.

The retinal photoreceptors of the eye of the American chameleon, Anolis carolinensis, have been considered to be exclusively cones. Its retina is unusual for possessing two foveas (areas associated with heightened visual acuity), with the major, central fovea deeply incised and very densely packed with photoreceptors. Immunoblotting and light- and electron microscopic-immunocytochemistry, using several opsin monoclonal antibodies previously found specific for rods, demonstrated the presence and localization of this protein in the Anolis retina. This visual pigment appears sparsely in a subpopulation of photoreceptors in the periphery but overwhelmingly in the central fovea. Complementary results with cone-specific antibody and lectin binding corroborated this spatial organization. These results, as well as those with geckos, suggest that photoreceptor morphology is not an accurate guide among the lacertilians to visual pigment content, and that this phylogenetic grouping may constitute a crossroads in vertebrate photoreceptor evolution.

Mapping cone photopigment optical density.

The distribution of cone photopigment across the retina affects the amount of light captured by cones at each retinal location. Cone photopigment optical density is measured in two ways, with reflectometry and/or with color matching. Color matching measures a higher optical density than does reflectometry. Control experiments confirm that large-field color matches measure photopigment optical density toward their outer edge. There is qualitative agreement as to photopigment distribution from both techniques near the fovea. Beyond 1 deg, color matching indicates little decrease in photopigment with increasing eccentricity, whereas retinal densitometry shows a steep decline in photopigment. The decrease in perifoveal optical density measured with reflectometry is attributed to the decrease in cone coverage from fovea to perifovea as rods and interphotoreceptor spaces increase. Differences among subjects in photopigment distribution near the fovea, measured with both techniques, reflect differences in the specialization of the foveal center for cone length and/or photopigment concentration per cone, which are factors influencing results from both techniques.

Interphotoreceptor retinoid-binding protein in the Golgi apparatus of monkey foveal cones. Electron microscopic immunocytochemical localization.

The presence of interphotoreceptor retinoid-binding protein (IRBP) in the Golgi apparatus of monkey cone photoreceptors was examined by electron microscopic immunocytochemistry. Intracellular labeling for IRBP was light to moderate over the Golgi complex of foveal and peripheral cones in the retinas of all four monkeys examined. Composite drawings of two to seven serial sections of inner segments indicated that the highest density of intracellular labeling occurred over the Golgi apparatus. The labeling density over the Golgi complex was 8.5-29-fold higher than that over a comparable area of inner segment cytoplasm. The presence of immunolabel for IRBP in the Golgi apparatus supported the findings of in situ hybridization studies that cone photoreceptors can synthesize IRBP.