Abstract
Purpose: :
The goal of this study was to three dimensionally assess the structural components of the ONH in normal human eyes.
Methods: :
Autopsy eyes (n=8) were obtained within 36 hours of preservation, fixed in 3% paraformaldehyde, and the ONH and surrounding sclera dissected from the globe. The tissue was embedded in agarose and sectioned normal to the ONH at 200 micron intervals using a Vibratome. Sections were imaged using a Zeiss 510 Meta SLM equipped with a Ti:Sapphire laser tuned to 820nm. Second harmonic generated signals (SHG, 410nm) and two photon excited fluorescence (TPEF, 500nm) images were collected with a lateral resolution of 0.44um at 2um intervals. Using an automated stage, a series of Z stacks were collected to encompass the entire ONH. The individual stacks were concatenated using Image J to create a single 3D data set. These sets were then segmented to highlight collagen (SHG) and elastin fibers (TPEF). Sections containing the lamina cribrosa were analyzed as mean projections for the dominant fiber orientation for both collagen and elastin within a grid placed over the image.
Results: :
The data demonstrated the intricate pattern of collagen and elastin fibers within the tissue. Clearly apparent was the circumferential ring of collagen surrounding the canal opening. Additionally, SHG demonstrated an interweaving of collagen fibers; anchoring laminar beams peripherally into the sclera and centrally into the central retinal vessels. The elastin fiber orientation followed that of the collagen beams within the peripheral sclera (r=0.71, P=0.001) and the lamina proper (r=0.44, P=0.001). However, at the canal border, elastin fibers were rotated approximately 45O from the collagen fibers. Reconstructions of these fibers demonstrated an arrangement of elastin with bundles of fibers parallel to the canal and fibers which appear to weave around the circumferential collagen fibers similar to an overcast stitch.
Conclusions: :
Vibratome sectioning of the ONH coupled with NLO optical sectioning provide high resolution 3D visualizations of the collagen and elastin structural organization in the human ONH. To our knowledge these data represent the first evidence of elastic columns and woven coils at the canal border. Additionally, these data emphasize the radial insertion of collagen beams both into the sclera and the central retinal vessels.
Keywords: extracellular matrix • optic nerve • imaging/image analysis: non-clinical