The superior pole of the eyes was marked with an electrical cautery without perforating the cornea. Eyes were carefully enucleated avoiding traction on the optic nerves. Right eyes and left eyes were separately fixed overnight at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The fixative was then replaced with 0.4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 and stored at 4°C.
57 Fixed eyes were conventionally dehydrated in graded ethyl alcohols. Alcohol was then substituted with xylene and the eyes embedded in paraffin. Serial sections (8 μm thick) through the optic nerve head were obtained maintaining the plane of cut parallel to the optical axis of the eye. Sections were stained with hematoxylin-eosin, bright-field imaged with an upright fluorescence microscope (BX51; Olympus, Melville, NY), and photographed (Qicam 12-bit Fast model 1394 camera; QImaging, Surrey, BC, Canada). Digital images were analyzed on computer (ImageJ 1.36b freeware available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), to measure the thickness of the RNFL at the boundary between retina and optic nerve
(Fig. 1) . The two sections at which the optic nerve had maximum size were selected for measurements. Overall, four RNFL thicknesses per optic nerve head section (section 1: superior, inferior; section 2: superior, inferior) were measured and averaged to obtain a single entry. RNFL thicknesses of eyes reaching the PERG endpoint were compared with average RNFL thicknesses of independent groups of 2-month-old DBA/2J mice (
n = 5) and 16-month-old mice (
n = 5). To account for interocular differences, RNFL data have been analyzed separately for eyes with worse PERG amplitude (first eye) and better PERG amplitude (second eye) at the endpoint. Since the number of axons entering the optic nerve is equal to the number of RGCs, RNFL thickness measurements in a given optic nerve head sector represents a specific index of both RGC and axon number originating from the corresponding retinal sector. In principle, for a given RNFL thickness the number of axons/RGCs could be calculated if axon density/diameter at the entrance of the optic nerve head were known. An approximate estimate of RNFL axon density might be obtained from axon densities reported for the retrobulbar portion of the optic nerve.
25 58 59 However, evaluation of the absolute number of axons from RNFL measurements may yield to ambiguous results since the RNFL becomes thicker as it approaches the optic nerve due to stratification of nerve fibers originating from different retinal eccentricities. RNFL thickness may be reduced due to either glaucomatous axon loss or increased distance from the optic nerve head. To avoid this uncertainty, we analyzed relative changes of RNFL thickness at a fixed optic nerve head location between mice of different ages. It was shown that relative changes in RNFL thickness in DBA/2J mice of different ages are in good agreement with corresponding changes of optic nerve axons.
20 60 We focused on RNFL thickness measurements, rather than axon/RGC counts, for the potential advantages offered by noninvasive RNFL measurements obtained with imaging techniques such as optical coherence tomography (OCT) that are now available for the mouse (Ruggeri M,
IOVS 2007;48:ARVO E-Abstract 1199).
61 Longitudinal evaluation of RGC function and RNFL thickness using PERG and OCT, respectively, represent powerful tools for neuroprotection studies of mouse glaucoma models. It should take into account, however, that RNFL thickness includes nonaxonal elements (i.e., glia, vessels) whose relative contribution may increase with increasing severity of glaucoma due to progressive axonal loss.
62 It is also possible that gliosis may replace axons,
63 64 65 66 and show a thicker than expected residual RNFL thickness in advanced stages of glaucoma, where few RGC axons are expected to be present.