Abstract
Purpose: :
The goal of this investigation is to identify the changes in the collagen structure of the LC subjected to elevation of intra-ocular pressures (IOP).
Methods: :
Four pairs of eyes were acquired from the San Diego Eye Bank within 36h from the time of autopsy. Pairs of eyes from the same individual were cannulated and one eye was maintained at 15mmHg and the contralateral eye maintained at 50mmHg pressure. The eyes were then fixed under pressure by perfusion with 3.2% paraformaldehyde in PBS. Following fixation, the optic nerve head (ONH) and surrounding sclera, ~4X4 mm of tissue, was excised, dehydrated with ethanol and infiltrated with LR-White resin. The tissue was then polymerized using a Pelco Biowave Pro microwave and the tissue block serially sectioned. Each 2um section was collected onto slides and then imaged using a Zeiss 510 Meta microscope. Second harmonic generation (SHG) from the tissue was accomplished using a two-photon 820nm femtosecond Coherent Chameleon Laser. SHG signal was collected from 410±20nm.ImageJ was used to threshold the images. Amira 5.4.0 was used to align the z-stack, and render the image in 3-D. The anterior laminar surface of each eye was landmarked using Amira and the Landmark module. From the landmarked surfaces, a 3-D surface was calculated and the surface area was extrapolated and recorded.
Results: :
SHG signals demonstrated the fibrillar collagen structure within ONH. This method allowed visualization of the laminar beams traversing the scleral canal opening and facilitated demarcation of the anterior laminar surface. When pairs of eyes fixed at 2 distinct pressures were compared, those fixed at 15mm Hg showed a surface area of 2.95 +/- 0.07 mm2 while those fixed at 50mm Hg showed a significant increase in surface area (3.38+/-0.05 mm2, P<0.05). Conversely, pairs of eyes fixed at the same pressure showed a surface area variance of only approximately 4 percent.
Conclusions: :
Increasing IOP leads to an increase in the anterior surface area of the LC. Visualization of the 3D surface area demonstrates that these increases are accomplished by both expansion of the scleral canal and deformation of the laminar beams. Interestingly, both the canal expansion and the posterior deformation of the laminar beams are most pronounced in the superior and inferior quadrants with little change in the nasal and temporal quadrants. These observations correlate well with clinical and experimental studies of the lamina in glaucoma.
Keywords: extracellular matrix • optic nerve • imaging/image analysis: non-clinical