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The relationship between retinal nerve fiber layer (RNFL) birefringence (&Dgr;n) and neurotubule density (NTD, retinal
ganglion cell (RGC) neurotubules per unit RNFL area) was investigated by correlating measurements of these two
parameters in 1 eye of a healthy cynomolgus monkey. Phase retardation per unit depth (PR/UD, proportional to &Dgr;n) was
measured at 5.6-15o intervals around the optic nerve head (ONH) with an enhanced polarization-sensitive optical
coherence tomography (EPS-OCT) instrument. Transverse tissue sections containing 3 RGC nerve fiber bundles from
each peripapillary RNFL octant were imaged with a transmission electron microscope (TEM). Morphological
measurements taken in TEM images were used by a novel algorithm to estimate NTD. Registered PR/UD and NTD data
were then correlated using single- and multi-level models, yielding correlation coefficients in the range 0.49 ⩽ r ⩽ 0.61
(0.06 ⩽ P ⩽ 0.11). It was found that in order for the single-level correlation coefficient (r = 0.61) to be statistically
significant (P ⩽ 0.05) and powerful (Power ⩾ 80%), NTD measurements in at least 16, rather than 8, RNFL sectors were
needed. Interestingly, a single-level correlation coefficient of r = 0.81 (P = 0.01) was calculated between octant-averaged
PR/UD and RGC axoplasmic area (Ax, axon area less non-cytoskeletal organelle area) mode. Ax represents a
RGC axon's neurotubule-inhabitable area. Intuitively, a strong relationship should exist between Ax and neurotubule
number if neurotubules provide the primary structural support for RGC axons and structural requirements are the same
in all RGC axons. If this relationship exists, error resulting from NTD estimation methods or preservation artifacts may
have caused lower observed correlations of PR/UD with NTD than with Ax mode, and more accurate methods of
measuring in vivo NTD may be required to determine an accurate relationship between RNFL birefringence and NTD.
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