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Sarah M Baldivia, Alexander Levy, Rafael Grytz; Quantifying Collagen Remodeling Deformations of Tree Shrew Sclera in Organ Culture. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5531.
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© ARVO (1962-2015); The Authors (2016-present)
Increasing evidence from animal studies suggests that unknown scleral remodeling mechanisms underlie myopia development. We are proposing a new methodology to quantify scleral collagen remodeling deformations at the macro- (tissue level) and micro-scale (lamellar level) in organ culture.
Three normal juvenile tree shrew eyes were enucleated at 35±2 days after eye opening, cleaned, stained for 18 hours in 1uM CNA35-tdTomato, a collagen binding fluorescent protein, and cultured for 5 days at constant IOP (15 mmHg) using a custom built, portable bioreactor. On day 1 of organ culture, we photobleached square patterns into each fluorescently labeled sclera at low magnification (25x) and parallel grid lines into scleral lamellae at the high magnification (~60x) using a multiphoton microscope. Second-harmonic generation (SHG) imaging was used to confirm that no thermal damage had resulted from photobleaching. On culture days 2, 4, and 5, the photobleached patterns were reimaged and new patterns were photobleached at different areas to compensate for fading patterns. The grid patterns were manually delineated. Tissue strain rates were computed as the change in photobleached square dimensions from one culture day to the next. Lamellar strain rate rates were computed for each lamellae section as the change in distance between two photobleached grid lines.
The tissue strain rate significantly decreased over time: 1.79±1.41 %/day, 0.17±0.72 %/day, and -0.94±0.97 %/day on Days 2, 4 and 5, respectively (mean±standard deviation). The lamellar strain rate was not significantly different between days: 0.03±3.74%, 0.94±5.26%, and -0.56±6.22% on Days 2, 4, and 5, respectively. The high variability in lamellar strain suggests that different lamellar sections elongated and others shortened. SHG showed no visible damage of the collagen micro-structure due to photobleaching.
Results suggest that scleral remodeling occurs in normal juvenile tree shrews. At the macro-scale, remodeling deformations continued initially but ceased and reversed with prolonged culture time, possibly due to loss of retinal signaling. Intra-lamellae micro-deformations may underlie the observed high variability in lamellar strains. This is the first study that quantified remodeling deformations in scleral shells. These tools can be applied to sclera from myopic eyes to decipher the scleral remodeling mechanisms in myopia.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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