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Objective To evaluate the appearance of the optic nerve head and lamina cribrosa in patients with glaucoma using spectral/Fourier-domain optical coherence tomography (SD-OCT) and to test for a correlation between lamina cribrosa thickness measured on SD-OCT images and visual field loss. Design Observational case series. Participants We evaluated 52 eyes of 30 patients with glaucoma or ocular hypertension. Methods The high-speed SD-OCT equipment used was a prototype system developed for 3-dimensional (3D) imaging. It had a sensitivity of 98 decibels (dB), a tissue axial resolution of 4.3 μm, and an acquisition rate of ∼18,700 axial scans per second. For 3D analyses, a raster scan protocol of 256 × 256 axial scans covering a 2.8 × 2.8 mm disc area was used. Lamina cribrosa thickness was measured on 3D images using 3D image processing software. Correlation between lamina cribrosa thickness and mean deviation (MD) values obtained using static automatic perimetry were tested for statistical significance. Main Outcome Measures Clarity of lamina cribrosa features, lamina cribrosa thickness, and MD values on static automatic perimetry. Results On 3D images, the lamina cribrosa appeared clearly as a highly reflective plate that was bowed posteriorly and contained many circular areas of low reflectivity. The dots of low reflectivity visible just beneath the anterior surface of the lamina cribrosa in en face cross-sections corresponded with dots representing lamina pores in color fundus photographs. The mean (±1 standard deviation) thickness of the lamina cribrosa was 190.5±52.7 μm (range, 80.5–329.0). Spearman rank testing and linear regression analysis showed that lamina cribrosa thickness correlated significantly with MD (Spearman σ = 0.744; P <0.001; r2 = 0.493; P <0.001). Different observers performed measurements of the lamina cribrosa thickness in SD-OCT cross-sectional images with high reproducibility (intraclass correlation coefficient = 0.784). Conclusions These 3D SD-OCT imaging clearly demonstrated the 3D structure of the lamina cribrosa and allowed measurement of its thickness, which correlated significantly with visual field loss, in living patients with glaucoma. This noninvasive imaging technique should facilitate investigations of structural changes in the optic nerve head lamina cribrosa in eyes with optic nerve damage due to glaucoma. Financial Disclosure(s) The authors have no proprietary or commercial interest in any materials discussed in this article.