Retinal Nerve Fiber Layer Reflectance Research Articles

Interpreting Retinal Nerve Fiber Layer Reflectance Defects Based on Presence of Retinal Nerve Fiber Bundles.

SIGNIFICANCEAdaptive-optics scanning-laser-ophthalmoscopy (AOSLO) retinal imaging of the retinal nerve fiber layer (RNFL) helps predict the severity of perimetric damage based on absence of fibers and projection of the defects in en face images of the RNFL from spectral-domain optical coherence tomography (SD-OCT).PURPOSEEn face images of the RNFL reveal reflectance defects in patients with glaucoma and predict locations of perimetric defects. These defects could arise from either loss of retinal nerve fiber bundles or reduced bundle reflectance. This study used AOSLO to assess presence of bundles in areas with RNFL reflectance defects on SD-OCT.METHODSAdaptive-optics scanning laser ophthalmoscopy was used to image a vertical strip of RNFL measuring approximately 30 × 3° between the optic disc and the fovea. Fifteen patients with glaucoma who had SD-OCT reflectance defects that passed through this region were chosen. Four patients had reflectance defects in both superior and inferior hemifields, so presence of bundles on AOSLO was assessed for 19 hemifields. Where bundles were present, the hemifield was scored for whether bundles seemed unusual (low contrast and/or low density). Perimetric defects were considered deep when sensitivity was below 15 dB.RESULTSTen hemifields had a region with no fibers present on AOSLO; all had a corresponding deep perimetric defect. The other nine hemifields had no region in the AOSLO image without fibers: four with normal fibers and five with unusual fibers. The only one of these nine hemifields with a deep perimetric defect was one with low-contrast fibers and overall thin RNFL.CONCLUSIONSRetinal nerve fiber layer reflectance defects, which were associated with deep perimetric defects, usually had a region with absence of fibers on AOSLO images of RNFL. Ability to predict severity of perimetric damage from en face SD-OCT RNFL reflectance images could benefit from quantification that differentiated between absence of fibers and unusual fibers.

Relief of Cystoid Macular Edema-Induced Focal Axonal Compression with Anti-Vascular Endothelial Growth Factor Treatment.

PurposeTo evaluate the mechanical compression of retinal nerve fiber layer (RNFL) by intraretinal cysts in macular edema and its relief with anti-vascular endothelial growth factor (anti-VEGF) treatment.MethodsOptical coherence tomography scans were used to measure RNFL thickness and reflectance at seven preselected points at and around the peak of the edema before and after anti-VEGF treatment in 10 patients (11 eyes) with branch retina vein occlusion (BRVO) and diabetic macular edema (DME). Scans through nonedematous retina and from the fellow eyes were taken as controls. Correlations were sought between the changes in retinal and RNFL thickness, RNFL reflectance, and the size of the intraretinal cysts.ResultsPostinjection RNFL thickness decreased significantly only at peak point of the edema (18.1 ± 2.7 vs. 13.8 ± 1.2 µm; P = 0.038), at its nasal edge (20.1 ± 2.7 vs. 15.5 ± 1.4 µm; P = 0.026), and 500 µm away from its nasal border (35.7 ± 6.0 vs. 20.1 ± 2.7 µm; P = 0.006) suggesting focal stagnation of the axoplasmic flow owing to compression at its peak point. Significant postinjection decreases in RNFL reflectivity were also noted at peak point of the cyst (164.9 ± 10.3 vs. 141.5 ± 12.6 arbitrary units [AU]; P = 0.037), at its nasal edge (166.8 ± 7.8 vs. 135.1 ± 10.2 AU; P = 0.02), and 1500 µm away from temporal edge (160.2 ± 6.2 vs. 141.1 ± 6.4 AU; P = 0.022). Cyst proximity to RNFL (D50 = 50 µm) was the only determinant significantly affecting the magnitude of the RNFL thickness change after anti-VEGF treatment (P = 0.001).ConclusionsIntraretinal cysts due to BRVO and DME locally compress overlying axons and induce anatomic changes suggestive of axoplasmic stagnation. This compression can be relieved with anti-VEGF treatment.Translational RelevanceFocal compression of RFNL by retinal cysts may indicate a need for early treatment of macular edema to prevent axonal loss, especially in patients with low axonal reserve

Temporal change of retinal nerve fiber layer reflectance speckle in normal and hypertensive retinas

This study investigated temporal change of retinal nerve fiber layer (RNFL) reflectance speckle in retinas with ocular hypertensive (OHT) damage and in control retinas from untreated eyes. Experimental OHT damage to rat retinas was induced by laser photocoagulation of the trabecular meshwork. A series of 660 nm reflectance images was collected from isolated retinas at 10-sec intervals. Areas containing speckled texture were selected on nerve fiber bundles. Correlation coefficients between images with different imaging delays were calculated and plotted as a function of delay. To evaluate the temporal change of speckles, decay of correlation coefficients with time was fitted with an exponential function characterized by a time constant τ. Reflectance per unit thickness (σ) of the areas was also measured and low σ was used as a surrogate of OHT damage. Speckle phenomena occurred in the control RNFL and the RNFL with reduced σ. In the control retinas, τ and σ were nearly constant along bundles but differed significantly among bundles in the same retinas. Among the control retinas, σ was similar, whereas τ varied significantly. In the retinas with OHT damage (low σ) τ could be within, greater or lower than the range in controls. The parameters τ and σ provide independent assessment of the RNFL with OHT damage. Measurements of temporal change of RNFL reflectance speckle may offer a method for detecting functional abnormality of the RNFL.

Response of the Retinal Nerve Fiber Layer Reflectance and Thickness to Optic Nerve Crush.

PurposeTo study the effects of acute optic nerve damage on the reflectance of the retinal nerve fiber layer (RNFL) and to compare the time courses of changes of RNFL reflectance and thickness.MethodsA rat model of optic nerve crush (ONC) was compared with previously studied normal retinas. The reflectance and thickness of the RNFL were studied at 1 to 5 weeks after ONC. Reflectance spectra from 400 to 830 nm were measured for eyes with ONC, their contralateral untreated eyes, and eyes with sham surgery. Directional reflectance was studied by varying the angle of light incidence. RNFL thickness was measured by confocal microscopy.ResultsAfter ONC, the RNFL reflectance remained directional. At 1 week, RNFL reflectance decreased significantly at all wavelengths (P < 0.001), whereas there was no significant change in RNFL thickness (P = 0.739). At 2 weeks, both RNFL reflectance and thickness decreased significantly, and by 5 weeks they declined to approximately 40% and 30%, respectively, of the normal values. Although RNFL reflectance decreased at all wavelengths, there was a greater reduction at short wavelengths. Spectral shape at long wavelengths was similar to the normal. Some of these changes were also found in the contralateral untreated eyes, but none of these changes were found in eyes with sham surgery.ConclusionsDecrease of RNFL reflectance after ONC occurs prior to thinning of the RNFL and the decrease is more prominent at short wavelengths. Direct measurement of RNFL reflectance, especially at short wavelengths, may provide early detection of axonal damage.

Reflectance Spectrum and Birefringence of the Retinal Nerve Fiber Layer With Hypertensive Damage of Axonal Cytoskeleton.

PurposeGlaucoma damages the retinal nerve fiber layer (RNFL). This study used precise multimodal image registration to investigate the changes of the RNFL reflectance spectrum and birefringence in nerve fiber bundles with different degrees of axonal damage.MethodsThe reflectance spectrum of individual nerve fiber bundles in normal rats and rats with experimental glaucoma was measured from 400 to 830 nm and their birefringence was measured at 500 nm. Optical measurements of the same bundles were made at different distances from the optic nerve head (ONH). After the optical measurements, the axonal cytoskeleton of the RNFL was evaluated by confocal microscopy to assess the severity of cytoskeletal change.ResultsFor normal bundles, the shape of the RNFL reflectance spectrum and the value of RNFL birefringence did not change along bundles. In treated retinas, damage to the cytoskeleton varied within and across retinas. The damage in retinal sectors was subjectively graded from normal-looking to severe. Change of spectral shape occurred near the ONH in all sectors studied. This change became more prominent and occurred farther from the ONH with increased damage severity. In contrast, RNFL birefringence did not show change in normal-looking sectors, but decreased in sectors with mild and moderate damage. The birefringence of severely damaged sectors was either within or below the normal range.ConclusionsVarying degrees of cytoskeletal damage affect the RNFL reflectance spectrum and birefringence differently, supporting differences in the ultrastructural basis for the two optical properties. Both properties, however, may provide a means to detect disease and to estimate ultrastructural damage of the RNFL in glaucoma.

Changes in Retinal Nerve Fiber Layer Reflectance Intensity as a Predictor of Functional Progression in Glaucoma.

PurposeWe determined whether longitudinal changes in retinal nerve fiber layer (RNFL) reflectance provide useful prognostic information about longitudinal changes in function in glaucoma.MethodsThe reflectance intensity of each pixel within spectral-domain optical coherence tomography (SD-OCT) circle scans was extracted by custom software. A repeatability cohort comprising 53 eyes of 27 participants (average visual field mean deviation [MD] −1.65 dB) was tested five times within a few weeks. To minimize test–retest variability in their data, a reflectance intensity ratio was defined as the mean reflectance intensity of pixels within the RNFL divided by the mean between the RNFL and RPE. This was measured in a separate longitudinal cohort comprising 310 eyes of 205 participants tested eight times at 6-month intervals (average MD, −0.99 dB; median rate of change, −0.09 dB/y). The rate of change of this ratio, together with the rate of RNFL thinning, and their interaction, were used to predict the rate of change of MD.ResultsIn univariate analyses, the rate of RNFL thinning was predictive of the rate of MD change (P < 0.0001), but the rate of change of reflectance intensity ratio was not (P = 0.116). However, in a multivariable model, the interaction between these two rates significantly improved upon predictions of the rate of functional change made using RNFL thickness alone (P = 0.038).ConclusionsFor a given rate of RNFL thinning, a reduction in the RNFL reflectance intensity ratio is associated with more rapid functional deterioration. Incorporating SD-OCT reflectance information may improve the structure–function relation in glaucoma.

Retinal nerve fiber layer reflectometry must consider directional reflectance.

Recent studies reveal that measurements of retinal nerve fiber layer (RNFL) reflectance provide more sensitive detection of glaucomatous damage than RNFL thickness, but most do not consider directional reflectance of the RNFL, an important source of variability. This study quantitatively compared RNFL directional reflectance, represented by an angular spread function (ASF), measured at different scattering angles, different wavelengths and different distances from the optic nerve head (ONH) and for bundles with different thicknesses (T). An ASF was characterized by its amplitude (A) and width (W). Internal reflectance of a bundle was expressed as A/T. The study found that A varied significantly with scattering angle and wavelength and that A/T was different among bundles but constant along the same bundle, indicating that the internal structure of axons may vary among bundles but does not change with distance. This study also found that W was larger near the ONH and at longer wavelengths, but did not depend on scattering angle or T. Because a 4.3° change in incident angle can change reflected intensity by a factor of 2.7, accounting for directional reflectance should improve the accuracy and reproducibility of RNFL reflectance measurements.

Localized changes in retinal nerve fiber layer reflectance intensity are related to localized functional loss in glaucoma

PurposeReflectance within the retinal nerve fiber layer (RNFL) may change prior to, or concurrent with, RNFL thinning in glaucoma. We hypothesize that reductions in RNFL reflectance intensity may be observed by optical coherence tomography (OCT), and may provide useful additional information beyond RNFL thickness.MethodsParticipants enrolled in an ongoing longitudinal study of glaucomatous progression had peripapillary circle scans acquired using spectral‐domain OCT, and performed automated perimetry, every 6 months. Data were analyzed from the most recent 8 visits with reliable results, from 211 eyes of 143 individuals. For each of the 52 visual field locations, intensity ratio and RNFL thickness were calculated within a 30º sector centered at the average location where corresponding nerve fibers enter the disc. Intensity ratio was defined as the mean intensity of pixels within the delineated RNFL boundaries divided by the mean intensity of pixels between the outer RNFL boundary and Bruch's Membrane. Rates of localized change were defined as the rate of change within each sector/location, minus the rate of global change. A mixed effects model was used to predict the rate of localized functional change from the rates of localized thickness and intensity ratio change within the corresponding sector.ResultsIn a combined model, the rate of localized functional loss was predicted by both rate of thinning and the interaction between the rates of thinning and intensity ratio change (both p &lt; 0.0001). For a given rate of RNFL thinning, a more negative rate of intensity ratio change predicted more rapid loss of sensitivity.ConclusionsReduction of RNFL reflectance over time is associated with loss of sensitivity at corresponding locations. While these are early results, they suggest potential improvements to the interpretation and quantification of OCT scans.

Reflectance Speckle of Retinal Nerve Fiber Layer Reveals Axonal Activity

This study investigated the retinal nerve fiber layer (RNFL) reflectance speckle and tested the hypothesis that temporal change of RNFL speckle reveals axonal dynamic activity. RNFL reflectance speckle of isolated rat retinas was studied with monochromatic illumination. A series of reflectance images was collected every 5 seconds for approximately 15 minutes. Correlation coefficients (CC) of selected areas between a reference and subsequent images were calculated and plotted as a function of the time intervals between images. An exponential function fit to the time course was used to evaluate temporal change of speckle pattern. To relate temporal change of speckle to axonal activity, in vitro living retina perfused at a normal (34°C) and a lower (24°C) temperature, paraformaldehyde-fixed retina, and retina treated with microtubule depolymerization were used. RNFL reflectance was not uniform; rather nerve fiber bundles had a speckled texture that changed with time. In normally perfused retina, the time constant of the CC change was 0.56 ± 0.26 minutes. In retinas treated with lower temperature and microtubule depolymerization, the time constants increased by two to four times, indicating that the speckle pattern changed more slowly. The speckled texture in fixed retina was stationary. Fixation stops axonal activity; treatments with either lower temperature or microtubule depolymerization are known to decrease axonal transport. The results obtained in this study suggest that temporal change of RNFL speckle reveals structural change due to axonal activity. Assessment of RNFL reflectance speckle may offer a new means of evaluating axonal function.

Wavelength-Dependent Change of Retinal Nerve Fiber Layer Reflectance in Glaucomatous Retinas

Retinal nerve fiber layer (RNFL) reflectance is often used in optical methods for RNFL assessment in clinical diagnosis of glaucoma, yet little is known about the reflectance property of the RNFL under the development of glaucoma. This study measured the changes in RNFL reflectance spectra that occurred in retinal nerve fiber bundles with different degrees of glaucomatous damage. A rat model of glaucoma with laser photocoagulation of trabecular meshwork was used. Reflectance of the RNFL in an isolated retina was measured at wavelengths of 400-830 nm. Cytostructural distribution of the bundles measured optically was evaluated by confocal imaging of immunohistochemistry staining of cytoskeletal components, F-actin, microtubules, and neurofilaments. RNFL reflectance spectra were studied in bundles with normal-looking appearance, early F-actin distortion, and apparent damage of all cytoskeletal components. Changes of RNFL reflectance spectra were studied at different radii (0.22, 0.33, and 0.44 mm) from the optic nerve head (ONH). Bundles in 30 control retinas and 41 glaucomatous retinas were examined. In normal retinas, reflectance spectra were similar along the same bundles. In glaucomatous retinas, reflectance spectra changed along bundles with the spectra becoming flatter as bundle areas approached the ONH. Elevation of intraocular pressure (IOP) causes nonuniform changes in RNFL reflectance across wavelengths. Changes of reflectance spectra occur early in bundles near the ONH and prior to apparent cytoskeletal distortion. Using the ratio of RNFL reflectance measured at different wavelengths can provide early and sensitive detection of glaucomatous damage.

Thickness, Phase Retardation, Birefringence, and Reflectance of the Retinal Nerve Fiber Layer in Normal and Glaucomatous Non-Human Primates

We identified candidate optical coherence tomography (OCT) markers for early glaucoma diagnosis. Time variation of retinal nerve fiber layer (RNFL) thickness, phase retardation, birefringence, and reflectance using polarization sensitive optical coherence tomography (PS-OCT) were measured in three non-human primates with induced glaucoma in one eye. We characterized time variation of RNFL thickness, phase retardation, birefringence, and reflectance with elevated intraocular pressure (IOP). One eye of each of three non-human primates was laser treated to increase IOP. Each primate was followed for a 30-week period. PS-OCT measurements were recorded at weekly intervals. Reflectance index (RI) is introduced to characterize RNFL reflectance. Associations between elevated IOP and RNFL thickness, phase retardation, birefringence, and reflectance were characterized in seven regions (entire retina, inner and outer rings, and nasal, temporal, superior and inferior quadrants) by linear and non-linear mixed-effects models. Elevated IOP was achieved in three non-human primate eyes with an average increase of 13 mm Hg over the study period. Elevated IOP was associated with decreased RNFL thickness in the nasal region (P = 0.0002), decreased RNFL phase retardation in the superior (P = 0.046) and inferior (P = 0.021) regions, decreased RNFL birefringence in the nasal (P = 0.002) and inferior (P = 0.029) regions, and loss of RNFL reflectance in the outer rings (P = 0.018). When averaged over the entire retinal area, only RNFL reflectance showed a significant decrease (P = 0.028). Of the measured parameters, decreased RNFL reflectance was the most robust correlate with glaucomatous damage. Candidate cellular mechanisms are considered for decreased RNFL reflectance, including mitochondrial dysfunction and retinal ganglion cell apoptosis.

Microtubule Contribution to the Reflectance of the Retinal Nerve Fiber Layer

The reflectance of the retinal nerve fiber layer (RNFL) arises from cylindrical light scattering elements distributed throughout its thickness. To determine whether these elements include axonal microtubules, eyecup preparations of the toad Bufo marinus were treated with the microtubule depolymerizing agent colchicine.Some eyecups were incubated for various times in different colchicine concentrations and then fixed and prepared for electron microscopy (EM). In other eyecups the reflectance of the RNFL at 440 nm was measured by means of imaging microreflectometry. After a period of baseline measurements, the bathing solution was changed either to one of identical composition (control experiments) or to one containing 10 mM colchicine. Measurements then continued for 2 to 3 hours.Quantitative EM showed that colchicine caused the density of axonal microtubules to decrease by approximately one third in 2 hours. Reflectometry showed, in control experiments, that the RNFL reflectance remained near baseline for the duration of an experiment. In contrast, after changing to colchicine solution, the RNFL reflectance declined to a level 37% to 55% below baseline.Microtubules made a major contribution to the 440-nm reflectance of the unmyelinated axons of the toad RNFL and could make a similar contribution to the reflectance of human RNFL.

Diattenuation and polarization preservation of retinal nerve fiber layer reflectance.

The diattenuation spectrum of the retinal nerve fiber layer (RNFL) reflectance has been predicted to depend strongly on the relative refractive index (m) of light-scattering cylinders. To constrain the values of m, diattenuation of the RNFL reflectance of isolated rat retina was measured with a multispectral imaging micropolarimeter. The RNFL reflection has very weak intrinsic diattenuation at all wavelengths (400-830 nm), which rejects all values of m > or = 1.03. Degree of polarization (DOP) for reflection from the RNFL was also measured. DOP was close to unity at all wavelengths, which indicates that the RNFL is a polarization-preserving reflector.

Theoretical model of the polarization properties of the retinal nerve fiber layer in reflection

In several optical technologies for glaucoma diagnosis, polarized light is used to assess the retinal nerve fiber layer (RNFL) of the eye. For better understanding of the polarization properties of the RNFL, it was modeled as a thick birefringent slab containing parallel light-scattering cylinders, and the Mueller matrix for reflectance was derived. The model predicts that (1) the RNFL reflectance has weak intrinsic diattenuation; (2) the diattenuation spectrum depends strongly on the relative refractive indices of the cylinders; (3) both scattering and birefringence contribute to retardation; and (4) the RNFL reflectance generally preserves polarization, but depolarization may be detectable for thick RNFL at short wavelengths.

Computerized densitometric measurement system (CDMS) for the quantitative analysis of diffuse retinal nerve fiber layer atrophy

Since observable retinal nerve fiber layer (RNFL) atrophy occures several years before irreversible glaucomatous visual field damage in glaucoma patients, quantitative assessment of the diffuse RNFL atrophy has been the goal of many research efforts. The loss of RNFL reflectance is informative sign of the diffuse RNFL atrophy, and appears in monochrome photograph of RNFL. We have, therefore, made a system by which we could measure the loss of RNFL reflectance and named it Computerized Densitometric Measurement System (CDMS). In this method, density profiles concentric with optical disc are extracted from the digitalized RNFL photograph. Density integral of superior and inferior segments in the density profile is used for the assessment of RNFL atrophy. Satisfactory results were obtained about the clinical utility of this method when the CDMS measurement was compared with the indicator of loss of visual function. Intra- and inter-operator reproducibility was also excellent.

Light scattering and form birefringence of parallel cylindrical arrays that represent cellular organelles of the retinal nerve fiber layer

The retinal nerve fiber layer (RNFL) comprises bundles of unmyelinated axons that run across the surface of the retina. The cylindrical organelles of the RNFL (axonal membranes, microtubules, neurofilaments, and mitochondria) as seen by electron microscopy were modeled as parallel cylindrical arrays in order to gain insight into their optical properties. Arrays of thin fibrils were used to represent organelles that are thin relative to wavelength, and the model took into account interference effects that may arise from spatial order. Angular and spectral light-scattering functions were calculated for the backscattering hemisphere. Scattering was much larger from axonal membranes than from microtubules or neurofilaments. Spectra from 400 to 700 nm show that scattering increases at shorter wavelengths for both axonal membranes and microtubules. At 560 nm, scattering from mitochondria modeled as thick cylinders was approximately the same as that from microtubules but showed little wavelength dependence. The model reveals differences in backscattered polarization ratios that may permit experimental discrimination between microtubule and membrane mechanisms for the RNFL reflectance. Calculated backscattering exceeds measured values by at least 1 order of magnitude, but calculated form birefringence for microtubule arrays is approximately the same as measured birefringence.