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Alexander Levy, Rafael Grytz; Quantifying Lamina Cribrosa Beam Strains in Tree Shrews using Two-Photon Fluorescence Microscopy. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
To quantify micro-scale strains of tree shrew lamina cribrosa (LC) beams induced by intra-ocular pressure (IOP) changes using a collagen type I specific fluorescent protein and two-photon fluorescence microscopy.
One adult tree shrew eye was mounted in a custom pressurization chamber after enucleation and stained for 24 hours with CNA35-tdTomato (Aper et al., PlosOne. 2015;9:e114983), a protein-based fluorescent probe for collagen imaging. The eye was preconditioned with 20 cycles from 5 to 45 mmHg. Parallel lines were photobleached into LC beams at 5mmHg using the laser of the two-photon microscope (Nikon A1 MP) and imaged at 5, 15, 30, and 45 mmHg IOP. The sclera was allowed to rest for 10 minutes after preconditioning and between pressure increases. Image intensity profiles along the LC beams were used to measure the distances between photobleached beam sections as the distance between one intensity valley to the next. LC beam strains were calculated using 5mmHg as the initial length.
The measured deformations were not significantly different from a normal distribution (p > 0.05 for each pressure, Shapiro-Wilk goodness-of-fit). LC beam strains were 1.11 ± 0.57%, 1.37 ± 0.62%, and 2.02 ± 1.04% (mean ± standard deviation) at 15, 30, and 45 mmHg, respectively. LC beam strains increased significantly from 15 to 30 and from 30 to 45 mmHg (p < 0.05, paired t-test).
A new photobleaching method was proposed to quantify IOP-dependent micro-scale strains in LC beam of tree shrews. The results show increased LC beam deformations with increasing IOP, suggesting a complex nonlinear deformation response in the one tested eye. The proposed method can provide new insight into the biomechanics of the LC and its potential role in glaucoma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
(a) Maximum intensity projection of CNA35-tdTomato labeled tree shrew LC showing a spoke-like beam architecture. (b) Box plot of the LC beam strains for IOP elevations from 5 mmHg to 15, 30, and 45 mmHg, showing a significant increase in strain for each pressure elevation (p < 0.05, paired t-test). (c-f) Zoomed in sections of red box in (a) showing laminar beams with photobleached lines and one representative length measure at 5 (c), 15 (d), 30 (e), and 45 mmHg (f) IOP. Scale bars: (a) 200μm (c-f) 50μm. * significant difference (p < 0.05)
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