Abstract
Purpose: :
To characterize the distributions of axon density and size and their inter-ocular symmetry in bilateral pairs of non-human primate optic nerves (ON) using an automated computer algorithm for counting 100% of the ON area and to further assess the method’s accuracy by comparing the size and density patterns at 3, 6, and 10 mm behind the eye in one normal ON.
Methods: :
Images covering 100% of the optic nerve cross-sectional area from 2 pairs of normal and 2 chronic ischemic (ET-induced, IOVS 2005; 46:3943-3956) optic nerves and their contralateral controls were captured at 100X using a light microscope outfitted with a computer-controlled X-Y-Z stage (BioQuant, Nashville TN; ASI, Eugene OR). Axon density, size, and total count from all captured images were then calculated using an automated computer algorithm (Reynaud et al, 3297/B311, ARVO 2007).
Results: :
In the normal ONs, average axon density ranged from 2.039x105 to 2.417x105 axons/mm2 and average axon size ranged from 0.414 to 0.578 µm2. The maximum inter-nerve differences within the normal ON pairs were 2.69% for density and 3% for average size. The chronic ischemic ONs showed no difference in density relative to their control eyes; however, there was an 8%-12% average axon size reduction and their axon size distributions had a consistent leftward shift as compared to their contralateral control eyes. In the normal ON studied at 3, 6 and 10mm behind the eye, the average axon sizes were 0.413, 0.452, and 0.481 µm2 respectively. While the axon density was different within the 3 sections due to the changing size of overall ON cross-sectional area, the total axon count at each location was consistent (1.156 x106 ± 0.007).
Conclusions: :
Pairs of normal optic nerves had symmetric axon size and density distributions. The chronic ischemic optic nerves, while having similar densities as their contralateral control eyes, showed a discernible difference in their axon size distributions. The normal ON with 100% axon counts at different distances from the ON head confirmed that size increases with distance from the eye and that the topography of axon size and density changes on route to the optic chiasm, while the total number of axons remains constant.
Keywords: optic nerve • image processing • microscopy: light/fluorescence/immunohistochemistry