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D. J. Brown, A. Lay, L. Lam, S. Said, J. Jester, D. S. Minckler; Quantitative Assessment of the Extracellular Matrix in the Optic Nerve Head. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5667.
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Our goal was to quantitatively assess the structural components of collagen and elastin in 3-dimensions across of the human optic nerve head (ONH).
Five pairs of eyes ranging in age from 59-84 were fixed in 4% paraformaldehyde in PBS overnight at 4°C. The ONH was then removed, embedded in low melting point agarose and then serially sectioned at 250 um using a Vibratome. Sections were collected as a continuous series and nonlinear optical images of two photon excited fluorescence (TPEF, elastin) and second harmonic generated signals (SHG, collagen) were acquired using a Zeiss 510 Meta LSM and Chameleon femtosecond laser and a 10X 0.3 NA objective. For each section, a total of 24 overlapping multi-plane images (512 x 512 pixels, 1.8 um lateral resolution) arranged in a grid (6 X4) were collected. Images from each section were then concatenated into a single Z-stack mosaic of 3072x2048 pixels X 125 um, 8-bit multichannel image file. Mosaics were then processed using a median filter (2x2 pixel) to reduce noise and normalized to match gray levels. The Z-stacks were then demarcated into regions, a threshold established to exclude background pixels and the percent area of included pixels recorded for each channel using Metamorph software (Molecular Dynamics). Statistical analyses were performed using Prism 4.0 (Graphpad).
We demonstrate that, as expected, the SHG signal co localized with the pattern of immunofluorescence achieved with an anti-collagen antibody. The TPEF signal correlated well with the pattern of immunofluorescence achieved with an anti-elastin antibody. Using the SHG to indicate collagen and TPEF to indicate elastin, the volume occupied by these matrix components varied considerably with position in the ONH. Significantly, the proportion of elastin relative to collagen was markedly higher in the prelaminar canal immediately anterior to the collagen beams of the lamina. This enrichment in elastin content was also noted in the tissues immediately adjacent to the canal wall.
The utilization of a multiphoton laser system allows one to obtain detailed views of the structural components of the ONH that is optically based and non-invasive. In addition, as the entire ONH can be assessed the data can be objectively and quantitatively analyzed to determine the microscopic density of collagen and elastin with the ONH.
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