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D. J. Brown, D. S. Minckler, J. V. Jester, L. Lam, C. Nien-Shy, A. Lay; High Resolution Three Dimensional Reconstruction of the Optic Nerve Head Using Second Harmonic Generation (SHG) and Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1191.
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This study was intended to digitally reconstruct the three-dimensional structure of the optic nerve head from human and various animal species commonly used in glaucoma research.
A series of post mortem human and animal eyes were obtained and embedded in low melting point agarose (10% W/V in PBS) and then sequential 300 micron sections were collected in coronal or transverse planes suing a Vibratome. Sequential sections were analyzed using an Axiovert 200 microscope (Zeiss, Jena, Germany), and imaged using a Plan ApoChromat 20x objective (NA = 0.75). Two-photon second-harmonic signals from collagen were generated with a mode-locked titanium:sapphire laser (Chameleon; Coherent Inc.). Selected sections were additionally stained with 0.5% DMSO, 0.5% Trition X-100, 2.5% Dextran 40 supplemented with 7U Alexa 543 phalloidin, 5 µM SYTO 59, and 1 µM Oregon Green paclitaxel (Molecular Probes, Invitrogen) to stain actin, nuclei, and tubulin, respectively. Confocal fluorescent signal detection was obtained by using the 488- and 543/633-nm laser lines of the argon and red helium-neon lasers, respectively. All samples were scanned using a 2-µm z-axis step size to generate 3-D data sets extending into the tissue to a depth of 200 µm.
Reconstructions of the data sets reveal that in human samples, the canal structure is a ring-like array of collagen that is periodically interspersed with hook-like collagen fibrils projecting into the rings. These hooked fibrils also define the anterior beginning of the laminar beams that cross into the canal opening. These features appear restricted to higher primates as these hooks were lacking in rabbit, pig, guinea pig, and canine samples though they did have ring-like arrays in the canal wall. Rat and mice were essentially lacking any distinct organization around or in the canal opening. Interestingly, in a single case of human glaucoma, the typical collagen organization was markedly disrupted and there was a notable absence of hook-like fibrils.
Distinct differences in collagen structure among various species may have implications in the choice of animal model in that rodents lack a well formed lamina and other species are lacking hook-like fibrils. These hook-like fibrils appear to suture the ring-like fibrils of the canal wall with the scleral matrix. This anatomic feature appears to be limited to higher primates and may be lost with glaucomatous damage.
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