Abstract
Purpose: :
Optical coherence tomography (OCT) is commonly used to obtain thickness measurements of the circumpapillary retinal nerve fiber layer (RNFL) in glaucoma, but the measurement does not give information regarding the composition of this region. Furthermore, it has been observed with histology that there is an increase in the glial content with glaucomatous damage which may confound the interpretation of the OCT measurements. The purpose of the present study was to quantify the neural and non-neural components of the retinal nerve fiber layer in both glaucomatous and control eyes.
Methods: :
Four sets of eyes from macaque monkeys with unilaterally induced glaucoma were evaluated with confocal imaging in the peripapillary RNFL. Fixed tissue from each eye was exposed to phalloidin-Alexa 488, DAPI, and anti-GFAP antibody to visualize actin in axons, nuclei, and astrocytes respectively. Segments of the RNFL corresponding to similar regions previously measured with OCT were examined using the Zeiss LSM510 Confocal Microscope (Zeiss Meditec, Dublin CA). RNFL thickness measures were made with the confocal images and compared to pre-mortem OCT measurements. The proportion of each stain in the RNFL was determined using a customized MATLAB program in order to evaluate the tissue compositon of the RNFL. Three to five comparisons at different cross sectional intervals were made from each corresponding region of the glaucomatous and control eye.
Results: :
Comparison of RNFL thickness measurements from the confocal images to those obtained from pre-mortem measurements using the OCT show agreement for all sets of eyes. Evaluation of cell bodies contained within the RNFL revealed no significant difference in the content of DAPI staining elements between the normal and glaucomatous retina (5-8% of total thickness). The degree of actin staining showed a significant decrease in the glaucomatous eyes versus the controls (50-82% versus 82-92% of total thickness). GFAP staining was elevated in the glaucomatous eyes (15-42% of total thickness compared to 2-30% in controls).
Conclusions: :
Thickness measurements from OCT correspond well with histological measurements performed with confocal microscopy. Eyes with experimental glaucoma show a significant decrease in the proportion of actin staining elements indicating a loss of ganglion cell axons, and an increase in the proportion of GFAP staining elements indicating an increase in glial tissue in the RNFL with glaucoma.
Keywords: nerve fiber layer • microscopy: confocal/tunneling • retinal glia