Relationship between birefringence and neurotubule density in the primate retinal nerve fiber layer

The relationship between retinal nerve fiber layer (RNFL) birefringence (Delta 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 Delta n) was measured at 5.6-15 degrees 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 (A(x), axon area less non-cytoskeletal organelle area) mode. A(x) represents a RGC axon's neurotubule-inhabitable area. Intuitively, a strong relationship should exist between A(x) 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 A(x) mode, and more accurate methods of measuring in vivo NTD may be required to determine an accurate relationship between RNFL birefringence and NTD.