Purchase this article with an account.
M. Winkler, C. Nien-Shy, B. E. Jester, S. Massei, D. S. Minckler, J. V. Jester, D. J. Brown; Quantitative Analysis of 3-D Reconstructions of Human ONH Using Multiphoton Imaging. Invest. Ophthalmol. Vis. Sci. 2009;50(13):310.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to quantify the distribution of collagen and elastin in the human optic nerve head (ONH) including the pre-laminar, laminar and post-laminar regions using array tomography and nonlinear optics.
Normal eyes from a 53 yo male and a 73 yo female were fixed in 4% PFA in PBS overnight at 4°C. The ONH was removed, embedded in LR White resin and serially sectioned at 2µm using a Leica Ultracut Ultramicrotome with a diamond knife. The sections formed continuous ribbons that were floated onto gelatin coated glass slides, maintaining orientation and correct order. Sections were then scanned individually using two photon excited fluorescence for elastin and second harmonic generated light for collagen with a Zeiss 510 Meta LSM and a Coherent Chameleon fs-laser. Reconstructions were generated using Amira software at a resolution of .91µm/pixel lateral and 2µm axial for a total image size of 3584x3584 pixels per plane. Images were thresholded to exclude background pixels. Positive pixel density per unit area was measured using Metamorph software.
The reconstruction shows a non-uniform distribution of collagen both in anterior-posterior direction as well as varying collagen distribution in each section. Mapping the collagen density by geographic region reveals significant differences in collagen content that result in "thin spots" with low collagen density as well as areas of very high collagen density. Additionally, TPEF imaging revealed a ring of elastin bordering the collagenous canal wall similar to that in arterial walls as well as a pad of elastic tissue just anterior to the collagenous laminar beams.
Our results demonstrate that these reconstructions can be objectively and quantitatively analyzed to determine the microscopic distribution of collagen and elastin within the ONH. The distribution of elastin could help explain the elastic response to normal changes in IOP associated with routine daily fluctuations and external forces exerted by the eyelid wiper or eye rubbing. The non-uniform distribution of collagen throughout the lamina cribrosa suggests a non-uniform response to an increase in IOP, which would help explain how nerve axons become damaged in glaucoma patients. The regions containing the least collagen seem to correspond with those areas of the ONH most susceptible to damage during the onset of glaucoma.
This PDF is available to Subscribers Only