**Purpose.**:
To determine the degree of correlation between spatial characteristics of the retinal nerve fiber layer (RNFL) birefringence (Δ*n* _{RNFL}) surrounding the optic nerve head (ONH) with the corresponding anatomy of retinal ganglion cell (RGC) axons and their respective organelles.

**Methods.**:
RNFL phase retardation per unit depth (*PR*/*UD*, proportional to Δ*n* _{RNFL}) was measured in two cynomolgus monkeys by enhanced polarization-sensitive optical coherence tomography (EPS-OCT). The monkeys were perfused with glutaraldehyde and the eyes were enucleated and prepared for transmission electron microscopy (TEM) histologic analysis. Morphologic measurements from TEM images were used to estimate neurotubule density (ρ_{RNFL}), axoplasmic area (*A* _{x}) mode, axon area (*A* _{a}) mode, slope (*u*) of the number of neurotubules versus axoplasmic area (neurotubule packing density), fractional area of axoplasm in the nerve fiber bundle (*f*), mitochondrial fractional area in the nerve fiber bundle (*x* _{m}), mitochondria-containing axon profile fraction (*m* _{p}), and length of axonal membrane profiles per unit of nerve fiber bundle area (*L* _{am}/*A* _{b}). Registered *PR*/*UD* and morphologic parameters from corresponding angular sections were then correlated by using Pearson's correlation and multilevel models.

**Results.**:
In one eye there was a statistically significant correlation between *PR*/*UD* and ρ_{RNFL} (*r* = 0.67, *P* = 0.005) and between *PR*/*UD* and neurotubule packing density (*r* = 0.70, *P* = 0.002). Correlation coefficients of *r* = 0.81 (*P* = 0.01) and *r* = 0.50 (*P* = 0.05) were observed between the *PR*/*UD* and *A* _{x} modes for each respective subject.

**Conclusions.**:
Neurotubules are the primary source of birefringence in the RNFL of the primate retina.

^{ 1 }Imaging devices have been developed to quantitatively measure RNFL thinning and birefringence loss. For example, optical coherence tomography (OCT) produces cross-sectional images of the retina allowing measurement of RNFL thickness.

^{ 2 }Polarization-sensitive OCT (PS-OCT)

^{ 3 }and scanning laser polarimetry (GDx; Carl Zeiss Meditec, Inc. Dublin, CA)

^{ 4 }measure phase retardation in the RNFL that arises from anisotropic structures in the RNFL.

^{ 5 }Changes in RNFL birefringence may precede RGC death.

*n*) is a dimensionless measure of the anisotropy of the refractive index (

*n*) of a material and is given by the difference between the extraordinary index (

*n*

_{e}) and ordinary index (

*n*

_{o}), where

*n*

_{o}and

*n*

_{e}are the refractive indices for polarization perpendicular and parallel to the axis of anisotropy respectively (Δ

*n*=

*n*

_{e}−

*n*

_{o}). Phase retardation is proportional to birefringence according to the expression Δ

*n*= λ

_{0}PR/360°Δ

*Z*where λ

_{0}is the free-space wavelength, PR is the phase retardation, and Δ

*Z*is the thickness of the material.

^{ 6 }In general, Δ

*n*of the RNFL is weakly wavelength dependent across visible and near infrared wavelengths.

^{ 7 }The RNFL demonstrates form birefringence that originates from anisotropic cylindrically shaped cellular structures in the RGC axons.

^{ 8,9 }The major cylindrical structures of the RGC axonal cytoskeleton are neurotubules (NT), neurofilaments, and neurotubule-associated proteins.

^{ 10 }Huang and Knighton

^{ 5 }identify neurotubules as the source of birefringence from the RNFL. Theoretical analysis has attributed RNFL reflectance to the cylindrically shaped mitochondria and axonal membranes.

^{ 9 }

^{ 11 }Neurotubule density (ρ

_{RNFL}) is defined in this report as a scalar estimate of the number of neurotubules per unit area of RNFL tissue (NT/μm

^{2}). There is strong evidence that the regional characteristic ρ

_{RNFL}surrounding the optic nerve head (ONH) is a source of birefringence,

^{ 5,12,13 }but it has not been clear whether ρ

_{RNFL}is the only source of birefringence.

^{ 9 }RNFL birefringence has been investigated as a possible diagnostic to detect early subcellular changes in glaucoma before there is any measurable change in RNFL thickness.

^{ 5,12,13 }Fortune et al.

^{ 13 }injected colchicine into the vitreous of nonhuman primate eyes and observed neurotubule disruption with a reduction of RNFL birefringence without any accompanying change in RNFL thickness. These results show that decreases in the number of neurotubules occur before thinning of the RNFL, and in situ measurement of ρ

_{RNFL}can be used as a diagnostic for cytoskeletal changes in RGC axons.

^{ 13 }In this study, we compared the measured birefringence signal in the peripapillary retina with the corresponding measured anatomic features to explore the origin of the RNFL birefringence.

*Z*

_{RNFL}) and single-pass phase retardation maps of the two cynomolgus monkeys were measured by using an enhanced polarization-sensitive optical coherence tomography (EPS-OCT) system described previously.

^{ 11,14 }EPS-OCT consists of a PS-OCT instrument combined with a nonlinear fitting algorithm to determine PR and Δ

*n*with high sensitivity in weakly birefringent tissues. The system utilizes a mode-locked Ti:Al

_{2}O

_{3}laser source (λ

_{0}= 830 nm, Δλ = 55 nm) linearly polarized at 45°. The

*Z*

_{RNFL}and PR maps are used to construct phase retardation per unit depth (

*PR*/

*UD*) area maps given in units of degrees of retardation per 100 μm of RNFL thickness (degrees/per 100 μm) by dividing local PR by

*Z*

_{RNFL}.

^{2}area. Ring scans are derived from sector scans spread 5° apart. The right eye of each subject was imaged twice on separate days, to assess reproducibility of retinal birefringence measurements.

*PR*/

*UD*sinusoid pattern

^{ 11,15,16 }surrounding the ONH was used to determine the angular interval around the ONH that RGC axon organelles must be sampled. Using the Nyquist criterion, RGC axon organelles were measured in eight angular sections (octants) of the peripapillary retina in the first eye sampled (1853 OS). Based on the correlation result between birefringence and ρ

_{RNFL}of the first eye, it was determined that 16 angular interval sections should be sampled around the ONH of the second eye (decreasing bundle variance within a region) to increase the power to 80% and detect a significant correlation between ρ

_{RNFL}and birefringence (Fig. 1). Each bundle within an angular section was chosen at random to eliminate bias. Nerve bundles were sampled from a 0.15-mm

^{2}rectangular area from each angular section, which contained approximately 15 to 20 nerve fiber bundles; therefore, 3 nerve fiber bundles approximate 15% to 20% and 7.5% to 10% of the sampled area of subjects 57204 and 1853, respectively. The exact number of bundles within each angular section was not known.

*A*

_{b}; Fig. 2).

*A*

_{a}), neurotubule number (

*k*), mitochondrial area (

*A*

_{m}), noncytoskeletal organelle areas (

*A*

_{o}), and unidentifiable features of the RNFL bundle. Neurotubules in each of the selected RGC axons were manually counted using the Cell Counter plug-in feature in Image J. A single person identified and counted the RGC organelles in subject 1853 and two different people identified and counted organelles for subject 57204.

^{ 17 }respectively. If RGC bodies were not present directly posterior to the nerve fiber bundle, then the posterior boundary was defined at the points were RGC axons were absent. A line that bisected Müller cell processes and their footplates at the ILM defined boundaries between adjacent nerve fiber bundles. Axons that appeared pale and had substantially fewer neurotubules were identified as “sparse” axons and were recorded apart from the “nonsparse” axons (Fig. 4). Amacrine cells or glial cells were not included in RGC axons counts when criteria described previously were used.

^{ 18 }

*A*

_{x}) measurements measured from TEM images were used in a statistical algorithm to estimate ρ

_{RNFL}. (

*A*

_{x}) was calculated as the difference between (

*A*

_{a}) and the total organelle area (

*A*

_{m}and

*A*

_{o}) for each RGC axon and was then plotted against the respective number of neurotubules (

*k*) in a scatter plot (Excel; Microsoft, Redmond, WA) to determine

*k*(

*A*

_{x}). Axoplasmic area probability distribution functions (

*p*[

*A*

_{x}]) were then computed for each bundle using a kernel estimation method based on an Epanechnikov kernel function. A Freedman-Diaconis bin width rule was used to determine the bandwidth of the kernel smoothing window and was adjusted using Scott's skewness factor. The axoplasmic fractional area in the bundle (

*f*

_{x}) is determined by summing all

*A*

_{x}measurements in the bundle and then dividing the result by the area of the bundle (

*A*

_{b}): Calculations of

*k*(

*A*

_{x}),

*p*(

*A*

_{x}), and

*f*

_{x}are used to estimate RNFL neurotubule density in the nonsparse axons of the nerve fiber bundle according to the expression: where ρ

_{RNFL}is given in units of number of neurotubules per unit RNFL area.

*k*(

*A*

_{x}) linear regression model. Individual estimates of neurotubule counts for each axon (ρ

_{x}) are weighted by the probability of a given

*A*

_{x}in the nerve fiber bundle (

*p*[

*A*

_{x}]

*dA*

_{x}) and by the fraction of axoplasmic areas in the bundle (

*f*

_{x}) which accounts for null ρ

_{RNFL}values in non-RGC areas of the RNFL bundle and effectively normalizes ρ

_{x}into ρ

_{RNFL}.

_{RNFL}in the temporal maculopapillary fibers (angular sections T

_{I}, T

_{SI}, and T

_{IS}) for both subjects. The

*A*

_{x}range for the nonsparse axons in this region was a narrow range and resulted in a low

*R*

^{2}in linear fits of

*k*versus

*A*

_{x}.

_{nsx}) from which a 25% neurotubule density threshold was set to classify sparse axons in the respective region. Once sparse axons were identified, a sparse axon neurotubule density (ρ

_{sx}) was determined. The ρ

_{nsx}and ρ

_{sx}were individually weighted by their respective

*f*

_{x}and summed to get an estimate for ρ

_{RNFL}in the respective section according to:

*PR*/

*UD*measurements falling within an angular section were averaged, resulting in an equal number of

*PR*/

*UD*values for every mean ρ

_{RNFL}. The averaged

*PR*/

*UD*measurements for subjects 1853 and 57204 resulted in ±22.5° and ±11.25° of angular error, respectively. Average

*PR*/

*UD*measurement positions were optimally registered with angular RGC axon organelle mean measurements to compute the correlation between them (Fig. 5). The shift was used to avoid

*PR*/

*UD*and ρ

_{RNFL}measurement positions taken at angular boundaries between sections in both subjects.

*PR*/

*UD*and RGC organelles. The first approach was a Pearson product moment correlation. Correlation coefficients and significance values were computed (SAS ver. 9; SAS, Cary, NC).

*PR*/

*UD*and RGC organelles to be correlated on two levels so that finer angular resolution of

*PR*/

*UD*measurements could be preserved. Multilevel modeling was implemented through the procedure PROC MIXED within the statistical analysis software. The model accounts for the random effect of angular position of

*PR*/

*UD*measurements.

*PR*/

*UD*maps were obtained by EPS-OCT from four eyes of two subjects. Results from both subjects are presented throughout the results section to demonstrate the similarities and differences in birefringence and morphologic measurements.

*PR*/

*UD*were relatively higher in the superior and inferior areas in both subjects (Fig. 6). Clusters of

*PR*/

*UD*radial area and ring scans within respective ρ

_{RNFL}angular section boundaries were averaged, resulting in an equal number of

*PR*/

*UD*measurements for every ρ

_{RNFL}value.

*PR*/

*UD*radial area and ring measurements are grouped into regional quadrants and averaged to give the mean

*PR*/

*UD*values listed in Table 1. One-way ANOVA was used to compare quadrant means. The temporal and inferior pair was the only quadrant pair that was not significantly different (

*P*< 0.05) for subject 1853. All quadrant pairs excluding the inferior and nasal pair were significantly different (

*P*< 0.05) for subject 57204.

Scan Pattern | 57204 OD | 1853 OS | ||||||||
---|---|---|---|---|---|---|---|---|---|---|

Radial Area Scan 1 | Radial Area Scan 2 | Ring Scan | Radial Area Scan | Ring Scan | ||||||

Mean | SD | Mean | SD | Mean | SD | Mean | SD | Mean | SD | |

Superior | 19.1 | 2 | 18.3 | 2.6 | 17.1 | 4.2 | 14.1 | 4.3 | 17.6 | 3.2 |

Inferior | 14.1 | 3.1 | 14.9 | 2.6 | 14.8 | 3.9 | 12.3 | 5.1 | 9.4 | 4.7 |

Temporal | 9.4 | 3.3 | 9.0 | 3.3 | 9.6 | 4.3 | 8.5 | 3.6 | 9.3 | 3.3 |

Nasal | 12.5 | 4.5 | 15.5 | 12.4 | 11.5 | 5.7 | 13.3 | 6.1 | 14.3 | 5.6 |

*PR*/UD measurement reproducibility was determined for both radial area scans and the ring scan of subject 57204 by calculating the standard error (SE)

^{ 15 }for each of the

*PR*/

*UD*measurements that were averaged within ρ

_{RNFL}angular section boundaries. To calculate the SE for each

*PR*/

*UD*measurement, the standard deviation (SD) of the cluster

*PR*/

*UD*measurements within angular section boundaries were divided by the square root of the number of clusters. The highest SE of all

*PR*/

*UD*measurements was ±7.1 deg/100 μm in the nasal region. The average SE of all three scans was ±1.9 deg/100 μm (Δ

*n*= 0.4 × 10

^{−4}) and was calculated by dividing the average SD of cluster

*PR*/

*UD*measurements from all three scans by the square root of the number of scans.

_{RNFL}for all bundles in both eyes are given in Tables 2 and 3 and are shown graphically in Figure 7A. Neurotubule densities were highest in the superior and inferior quadrants and lowest in the temporal and nasal regions of both subjects. The Pearson product moment correlation was used to find the similarity between the octant mean ρ

_{RNFL}between the two subjects (Fig. 7B). The result was a correlation coefficient of 0.97 (

*P*< 0.001).

Section | Bundle 1 | Bundle 2 | Bundle 3 | Mean | SD |
---|---|---|---|---|---|

N_{S} | 66.0 | 72.2 | 76.3 | 71.5 | 5.2 |

S_{N} | 110.1 | 69.8 | 64.6 | 81.5 | 24.9 |

S_{T} | 126.5 | 79.4 | 85.4 | 97.1 | 25.7 |

T_{S} | 69.9 | 64.7 | 69.6 | 68.1 | 2.9 |

T_{I} | 32.5 | 28.8 | 37.6 | 33.0 | 4.4 |

I_{T} | 80.5 | 96.3 | 69.9 | 82.2 | 13.3 |

I_{N} | 98.4 | 77.0 | 68.3 | 81.2 | 15.5 |

N_{I} | 49.6 | 74.6 | 90.1 | 71.4 | 20.4 |

Section | Bundle 1 | Bundle 2 | Bundle 3 | Mean | SD |
---|---|---|---|---|---|

N_{SI} | 53.7 | 42.6 | 37.1 | 44.5 | 8.5 |

N_{SS} | 66.9 | 54.2 | 36.1 | 52.4 | 15.5 |

S_{NL} | 77.0 | 45.1 | 68.4 | 63.5 | 16.5 |

S_{NM} | 87.7 | 66.9 | 68.3 | 74.3 | 11.6 |

S_{TM} | 45.1 | 59.8 | 45.8 | 50.2 | 8.3 |

S_{TL} | 63.0 | 18.1 | 60.8 | 47.3 | 25.3 |

T_{SS} | 33.0 | 26.2 | 33.5 | 30.9 | 4.1 |

T_{SI} | 4.5 | 9.3 | 33.1 | 15.6 | 15.3 |

T_{IS} | 11.3 | 5.6 | 3.7 | 6.9 | 3.9 |

T_{II} | 46.5 | 35.0 | 42.3 | 41.3 | 5.8 |

I_{TL} | 72.6 | 24.5 | 83.5 | 60.2 | 31.4 |

I_{TM} | 50.6 | 65.6 | 47.2 | 54.5 | 9.8 |

I_{NM} | 42.1 | 73.7 | 41.4 | 52.4 | 18.5 |

I_{NL} | 73.7 | 22.3 | 25.6 | 40.5 | 28.8 |

N_{II} | 18.4 | 73.0 | 76.7 | 56.0 | 32.6 |

N_{IS} | 17.7 | 42.0 | 38.8 | 32.9 | 13.2 |

*k*and

*A*

_{x}used in the ρ

_{RNFL}estimate was similar across all angular sections for both subjects (Fig. 8A). As axon size increased, the neurotubule number also increased, but the number of neurotubules per unit axon area decreased. This finding is similar to other studies investigating axon size and neurotubule density in the optic nerve head.

^{ 19 }

*P*= 0.005) between

*PR*/

*UD*and ρ

_{RNFL}(Table 4), and the multilevel model resulted in standardized regression coefficients of 0.50 (

*P*= 0.04). The averaged pattern of

*PR*/

*UD*is shown superimposed on respective ρ

_{RNFL}for both subjects in Figure 9.

Statistical Model | r | P |
---|---|---|

Pearson's correlation | 0.67 | 0.005 |

Multilevel model | 0.5 | 0.04 |

*A*

_{x}) mode, axon area (

*A*

_{a}) mode, slope (

*u*) of neurotubule number versus axoplasmic area (neurotubule packing density), fractional area of axoplasm in the nerve fiber bundle (

*f*

_{x}), mitochondrial fractional area in the nerve fiber bundle (

*x*

_{m}), mitochondria-containing axon profile fraction (

*m*

_{p}), and length of axonal membrane profiles per unit nerve fiber bundle (

*L*

_{am}/

*A*

_{b}).

*PR*/

*UD*are summarized in Table 5. Correlation coefficients of

*r*= 0.81 (

*P*= 0.01) and

*r*= 0.5 (

*P*= 0.05) were observed between

*PR*/

*UD*and mean

*A*

_{x}mode in subjects 1853 and 57204 (Fig. 10), respectively. Mean

*A*

_{x}mode values were similar in the superior and inferior portions of both eyes and were larger than temporal and nasal portions. Mean

*A*

_{a}and

*PR*/

*UD*yielded a significant correlation coefficient of

*r*= 0.79 (

*P*= 0.01) in subject 1853 (Fig. 10B).

RGC Axon Morphologic Measurement | Abbreviation | 57204 OD | 1853 OS | ||
---|---|---|---|---|---|

r | P | r | P | ||

Least-squares linear slope (number of NTs per unit A_{x} area), NT/μm^{2} | Mean u | 0.70* | 0.0028 | 0.68 | 0.0941 |

Mean RGC axoplasmic area mode, μm^{2} | Mean A _{x} | 0.50* | 0.0500 | 0.81* | 0.0145 |

Mean axon area mode, μm^{2} | Mean A _{a} | 0.47 | 0.0600 | 0.79* | 0.0184 |

Neurotubule density (number of NTs per unit RNFL area), NT/μm^{2} | Mean ρ_{RNFL} | 0.67* | 0.0050 | 0.61 | 0.1077 |

Axoplasmic fractional area (per unit RNFL bundle area), % | Mean f | 0.34 | 0.1924 | 0.51 | 0.1934 |

Mitochondrial fractional area (per unit RNFL bundle area), % | Mean x _{m} | 0.02 | 0.9500 | — | — |

Mitochondria-containing axon profile fraction (per unit RNFL bundle area), % | Mean m _{p} | — | — | 0.24 | 0.5708 |

Axonal membrane length (per unit RNFL bundle area), μm^{−1} | Mean L _{am}/A _{b} | 0.24 | 0.3670 | 0.23 | 0.5811 |

*A*

_{x}) mode, neurotubule (NT) number (

*k*) versus

*A*

_{x}least-squares linear slope (

*u*), and fractional area of axoplasm per unit nerve fiber bundle (

*f*

_{x}) measurements are associated with nonsparse axons only. Axon area (

*A*

_{a}) mode, mitochondrial fractional area (

*x*

_{m}), mitochondria-containing axon profile fraction (

*m*

_{p}), and length of axonal membrane per unit nerve fiber bundle area (

*L*

_{am}/

*A*

_{b}) measurements are associated with both sparse and nonsparse axons.

*r*= 0.7 (

*P*= 0.002) was observed between section averaged

*PR*/

*UD*and the

*k*versus

*A*

_{x}regression fit slope (

*u*) that is used as a measure of neurotubule packing density (Fig. 11). Arcuate bundle regions had the highest packing density, followed by the nasal region, and last the papillomacular region.

*PR*/

*UD*measurements were highest superior and inferior to the ONH for both subjects. In subject 57204, the average of the quadrant

*PR*/

*UD*values given in Table 1 for the superior and temporal region are 18.2 ± 1.0 deg/100 μm (Δ

*n*= 4.2 × 10

^{−4}) and 9.3 ± 0.3 deg/100 μm (Δ

*n*= 2.1 × 10

^{−4}). Cense et al.

^{ 15 }measured the RNFL birefringence of two human subjects in vivo using PS-OCT at 840 nm. They also reported higher RNFL birefringence (4.1 × 10

^{−4}) in the superior and inferior areas with the lowest in the temporal region (1.2 × 10

^{−4}). Huang et al.

^{ 16 }calculated similar results of averaged birefringence for the superior (4.2 × 10

^{−4}) and temporal region (2.5 × 10

^{−4}) of 12 human eyes. The SE was greatest in the nasal region for subject 57204 and may have resulted from a thinner RNFL area or eye movements. This finding was similar to others.

^{ 15 }

*PR*/

*UD*measurements comes from any error in the RNFL thickness (

*Z*

_{RFNL}) measured from EPS-OCT intensity images and phase retardation (PR) estimates from the EPS-OCT algorithm. Error in the

*Z*

_{RFNL}comes from the refractive index range for the RNFL (4%,

*n*= 1.34–1.39)

^{ 20 }and uncertainty in the exact location of the RNFL boundaries due to the axial resolution limit of the EPS-OCT instrument (5 μm). Noise creates an uncertainty of approximately ±1° for PR estimates. Thus, uncertainty in PR is fractionally greater in regions with low PR such as the nasal and temporal regions. Radial and rotational error in position of the eye during imaging may have caused a registration error in

*PR*/

*UD*measurements.

_{RNFL}determination include: sampling RNFL bundles near the angular section boundaries, misclassified cells or cellular structures, different observers for the two eyes studied, and sampling location from the ONH for each eye. The higher ρ

_{RNFL}values for subject 1853 can be explained by counter bias between the first and second eye. The mean difference (95% limits of agreement) between neurotubule counts of the same RGC axons of two counters for subject 57204 was −1.6(−8.8 to 5.6) neurotubules.

_{RNFL}within nerve bundles than others. Most of the variability could be attributed to differences in RNFL nerve bundle area (

*A*

_{b}) measurements, since ρ

_{RNFL}estimates are calculated using

*A*

_{b}. The standard deviation of RNFL thickness of human retina ranges from 15 μm in the temporal region to 26.5 μm in the inferior region.

^{ 21 }Standard deviations of ρ

_{RNFL}greater than ±20 NT/μm

^{2}occurred in the arcuate bundles and the bundles located in the inferior nasal portion of the RNFL near major blood vessels. Bundles lying close or adjacent to temporal blood vessels that approximately overlie the arcuate nerve bundles

^{ 22 }were not excluded from the sampled population. The few histologic studies of primate RNFL thickness

^{ 18,22–24 }did not measure thickness near large blood vessels because RNFL borders could not be accurately determined.

^{ 25 }The RNFL bundle area variation near and away from blood vessels as well judgment as to the location of the RNFL borders would produce variability in ρ

_{RNFL}estimates.

*A*

_{b}measurement. Ogden

^{ 22 }reported that the proportion of RNFL bundle area occupied by glia is independent of RNFL thickness and does not vary regularly with distance from the ONH in cynomolgus and rhesus monkeys. Angular sections with the highest variability in ρ

_{RNFL}estimates also had the largest variability in glial content percentage.

_{SI}, T

_{IS}, and T

_{I}are sampled from the papillomacular bundle fibers and have the smallest RGC fibers.

^{ 26 }A low mean ρ

_{RNFL}may be attributable to a combination of a thin RNFL

^{ 23 }and low number of RGC axons

^{ 27 }that have a significantly smaller mean neurotubule number. The sparse axons in the papillomacular bundles could be a fixation artifact if the smaller axons were not adequately preserved by the perfusion of fixative. A similar pattern of sparse axons was observed in the two eyes studied which should give some assurance that the pattern was not unique to a single eye; however, it is possible that the neurotubules in the small axons of the papillomacular bundle degenerated faster than the large axons found elsewhere. The neurotubules polymerize and depolymerize very quickly and the smaller axons may be more fragile than the larger ones. The sparse axons and fewer nonsparse axons within octant T

_{I}and angular sections T

_{SI}and T

_{IS}could also be attributed to bilateral optic atrophy (BOA).

^{ 28 }

_{NL}, S

_{NM}, S

_{TM,}I

_{TM}, and I

_{NM}which are sampled from arcuate fibers. The arcuate portions of the RNFL are frequently involved in glaucoma and optic neuropathies.

^{ 27 }Higher birefringence in the superior and inferior region of the eye can be attributed to a combination of higher neurotubule packing density, larger axons, and less compartmentalization by glial tissue than other regions of the eye.

^{ 27 }

*A*

_{x}and

*PR*/

*UD*in both eyes is interesting because axoplasmic regions of RGC axons are the onlyareas that neurotubules can inhabit. In addition, there was a significant correlation between

*A*

_{a}and

*PR*/

*UD*in subject 1853. These findings suggest that a structure within the axoplasm is the source of the birefringence signal. The mean (

*u*) (neurotubule packing density) was more significantly correlated with

*PR*/

*UD*than the mean ρ

_{RNFL}for subject 57204. This suggests that the birefringence surrounding the ONH results from a difference in NT packing density of the axons themselves and not from a difference in the packing density of axons within bundles.

*L*

_{am}/

*A*

_{b}failed to correlate with

*PR*/

*UD*in subject 57204, despite increased sampling and further supports the finding

^{ 29 }that cell membranes are not the source of birefringence. Mitochondrial fractional area (

*x*

_{m}) and the percentage of mitochondria-containing axon profiles (

*m*

_{p}) did not correlate with

*PR*/

*UD*for subject 57204 and 1853, respectively.

*n*and

*PR*/

*UD*(

*P*= 0.0028) for all the morphologic parameters studied was the slope (

*u*) of the regression fit of neurotubule number (

*k*) versus axoplasmic area (

*A*

_{x}), which represents the neurotubule packing density.