Wavelength-dependent change of retinal nerve fiber layer reflectance in glaucomatous retinas

Abstract

Purpose: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Figure 1.

Calculation of RNFL reflectance. Nerve fiber bundles appear as bright stripes and are separated by gaps—the darker space between bundles. The reflectance (R) of the defined bundle area (thick box) includes the reflectance of the bundle alone (R_bundle) and its underlying tissue (R_background). The reflectance of the bundle alone is calculated as R_bundle = R − R_background, where R_background is estimated from the average reflectance of nearby gap areas (thin boxes). Image at 440 nm.

Figure 2.

Axonal cytoskeleton in retinas with different degrees of glaucomatous damage. Retinas were stained simultaneously for F-actin, MTs, and NFs. (A) Normal-looking bundles with uniformly stained cytoskeletal components within the bundles. (B) Bundles with irregularly stained F-actin (thin white arrows) and nuclei (thick white arrows) embedded within the bundles. (C) Severely damaged bundles with apparent network (yellow arrows) of F-actin between bundles and distorted strands of F-actin, MTs, and NFs within bundles. Images taken with 40× oil objective. Arrow heads: blood vessels.

Figure 3.

Reflectance spectra of gap areas are similar in normal (black line) and glaucomatous (gray line) retinas. Solid lines: average spectra of all gap areas defined for the bundles analyzed in this study. Dashed lines: ± standard deviation. Reflectance value was calibrated with the diffuse white reflector (see text for details).

Figure 4.

RNFL reflectance spectra in a normal retina. (A) Reflectance image (440 nm) shows bundles as bright stripes against a darker background. Reflectance of bundle areas (box) was analyzed at radii of 0.22 (r_a), 0.33 (r_b), and 0.44 (r_c) mm from the center of the ONH (not shown). Arrow: blood vessel. (B) The RNFL reflectance spectra of the bundle areas defined in 4A. The reflectance decreases with increasing wavelength. The reflectance spectra are similar along the same bundle. Solid line: fitted spectrum with a two-mechanism scattering model with parameters n = 134 ± 38, s = 165 ± 14 nm, w = 141 ± 47 nm, and R² = 0.97. The inset shows the scattering model (see text for details).

Figure 5.

RNFL reflectance spectra of bundles with different degrees of cytoskeleton damage (bundle areas shown in Fig. 2). (A) Normal-looking cytoskeleton. The spectrum is similar to the control with slightly flat reflectance at short wavelengths. (B) Distorted F-actin. The spectrum has apparent change at short wavelengths. (C) Severely distorted cytoskeleton. The whole spectrum becomes flat. Symbols: measured reflectance. Solid line: fitted spectrum with a two-mechanism scattering model. I_ang and S_ang: incident and scattering angles, respectively. s, w, n: fitted parameters (see text for details). ρ_S and ρ_L: reflectance ratios calculated from measured data points. Reflectance spectra are normalized to the average reflectance at 500 to 560 nm and plotted on log-log coordinates.

Figure 6.

RNFL reflectance spectra along one bundle in a glaucomatous retina with normal-looking cytoskeleton. The spectra are different along the bundle. The slope of reflectance decrease at short wavelengths becomes shallower as the bundle area approaches the ONH. The whole spectrum looks flatter at r_a, while the spectrum at r_c is similar to that of normal. r_a = 0.22 mm, r_b = 0.33 mm, and r_c = 0.44 mm from the center of the ONH. Reflectance spectra are normalized to the average reflectance at 500 to 560 nm and plotted on log-log coordinates. I_ang, S_ang: incident and scattering angles.

Figure 7.

Quantitative comparisons of RNFL reflectance spectra. (A) Calculated reflectance ratios ρ_S and ρ_L. (B) Fitted parameters (s, w, n) of different groups at radii of 0.22 (r_a), 0.33 (r_b), and 0.44 (r_c) mm from the center of the ONH. s and w are measured in nanometers. n is the number of thin cylinders. G1: bundles with normal-looking cytoskeleton. G2: bundles with distorted F-actin. G3: bundles with severely distorted F-actin, MTs, and NFs. *Indicates statistically significant difference between the treated groups and the control.