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Yu-Qiang Soh, Shakil Rehman, C. J. R. Sheppard, Roger W. Beuerman; Three Dimensional Quantification of Lamina Cribrosa Pore Deformation With Second Harmonic Generation Imaging. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4981.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate second-harmonic-generation (SHG) microscopy as a modality for imaging lamina cribrosa (LC) pores in three dimensions, while the LC is deformed in an ex-vivo setting to simulate changes in intra-ocular pressure (IOP).
A freshly enucleated porcine eye was dissected to reveal the LC and peripapillary sclera, which was then fashioned into a circular disc 20mm in diameter, with the LC at the centre of the disc. The specimen disc was clamped on a custom-built microscope stage in which the extra-ocular aspect was exposed to atmospheric pressure, while the intra-ocular aspect formed a section of the inner wall of a fluid filled chamber, directly exposing the LC to a body of fluid whose pressure could be changed to simulate fluctuations in IOP. A water immersion microscope objective placed within the fluid chamber was then used to image the lamina cribrosa. Combining high-resolution 2-dimensional frontal-plane LC images with a sequence of fine optical sections of the LC obtained along the Z-axis, changes in the sizes of LC pores could be accurately assessed while taking into account the anterior-posterior movement of the LC when fluid pressure was changed. Eight LC pores were tracked as simulated IOP was changed from 15mmHg to 40mmHg, then back to 15mmHg, with 30 minutes of settling time allowed between each change of pressure. The changes in maximum and minimum pore diameters, pore area, pore circularity and LC surface inclination were measured and analysed.
When IOP was increased from 15mmHg to 40mmHg, the ranges of change of a) maximum and minimum pore diameters were -7.89% to +1.32%, and -6.51% to +11.44%; b) pore area was -10.35% to +11.13%; and c) pore circularity was -5.93% to +6.08%. Upon reversal of pressure back to 15mmHg, the ranges of change (values compared to the first set of baseline measurements at 15mmHg) of a) maximum and minimum pore diameters were -3.83% to +6.89%, and -9.08% to +4.26%; b) pore area was -7.33% to +6.07%; and c) pore circularity was -2.99% to +6.28%. Measurements of the anterior-posterior inclination of the LC surface at each pore revealed that when pressure was increased from 15mmHg to 40mmHg, there was a change in inclination within the range of -6.14 degrees to +5.59 degrees. Reversal of pressure to baseline induced a change within the range of +3.45 degrees to -3.41 degrees.
SHG imaging of the lamina cribrosa allowed precise measurements of changes in LC pore dimensions and inclination as IOP is varied. Pore dimensions failed to consistently return to basline values upon exposure to changes in IOP, suggesting hysteresis and possibly irreversible mechanical effects when IOP is changed.
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