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James Germann, Eduardo Martinez-Enriquez, Silvia Bueno, Ashik Mohamed, Sushma Nandyala, Sunita Chaurasia, Susana Marcos; Microscopic organization of corneal collagen: Comparison between keratoconic and normal human corneas. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2588.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus is an ocular disease that results in a weakening of the cornea on a macroscopic scale, but the microscopic changes that occur are still in question. By identifying a biomarker on the microscopic scale, medical intervention can be applied before sizeable macroscopic changes occur.
Donor human corneas (n=5) and keratoconic tissues from anterior lamellar keratoplasty surgery (n=5) were preserved in formaldehyde and imaged with a custom Second Harmonic Generation (SHG) microscope in the forward and backward directions (Germann et al, BOE 2017). In each sample, 6-15 z-stacks (4 µm vertical spacing, 150×150 μm en face) of images were taken in different lateral locations (0.5 mm apart). Image stacks with a mean forward-to-backward SHG ratio (F/B) >1 were selected for processing in order to remove excessively scattering tissue from analysis. Data were obtained for the anterior stroma in healthy and keratoconus specimens. Image stacks were then processed to quantify the divergence in collagen fiber direction by assigning an order coefficient (OC, range 0-1), with high values signifying a more uniform direction. The mean SHG signal per image was measured as a metric for collagen scattering.
Of the image stacks, 36/42 donor stacks and 30/34 keratoconus stacks had a mean F/B >1. Mean thickness of the analyzed keratoconic samples was 128±79 μm and the donor anterior stroma was 240±67 μm. Mean OC of donor and keratoconic image stacks was 0.412±0.014 and 0.351±0.011 respectively, with mean donor OC 8% larger (p<0.01, ANOVA). Mean SHG signal in the donor anterior stroma was 2.09±0.71 × 106 and 1.13±0.41 × 106 counts in the forward and backward directions respectively. In keratoconic samples, mean SHG signal was 1.39±0.85 × 106 and 0.54±0.33 × 106 counts in the forward and backward directions respectively. The mean F/B of donor and keratoconic samples was 1.96±0.55 and 2.67±0.94 respectively, with the keratoconic F/B 15.3% larger (p<0.01, ANOVA).
The keratoconic tissue had a more variable fiber direction (lower OC) than the anterior stroma of donor corneas and produced less SHG signal. The larger F/B also suggests a change in fiber diameter or lamellae spacing in keratoconus samples. These findings indicate that divergence of fiber orientation and average SHG signal in the anterior stroma can be useful biomarkers in differentiating keratoconus from healthy corneas.
This is a 2020 ARVO Annual Meeting abstract.
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