The Relationship Between Retinal Ganglion Cell Axon Constituents and Retinal Nerve Fiber Layer Birefringence in the Primate

The Relationship between Retinal Ganglion Cell Axon Constituents and Retinal Nerve Fiber Layer Birefringence in the Primate

Ginger M. Pocock,1,2 Roberto G. Aranibar,1 Nate J. Kemp,1 Charles S. Specht,3 Mia K. Markey,4 and H. Grady Rylander III1

PURPOSE. To determine the degree of correlation between spa- laucoma is a progressive optic nerve disease characterized tial characteristics of the retinal nerve fiber layer (RNFL) bire- Gby a loss of retinal ganglion cell (RGC) axons and thinning ⌬ fringence ( nRNFL) surrounding the optic nerve head (ONH) of the retinal nerve fiber layer (RNFL). Axon loss can be as with the corresponding anatomy of retinal ganglion cell (RGC) much as 50% before the disease is clinically detectable.1 Imag- axons and their respective organelles. ing devices have been developed to quantitatively measure RNFL thinning and birefringence loss. For example, optical METHODS. RNFL phase retardation per unit depth (PR/UD, proportional to ⌬n ) was measured in two cynomolgus coherence tomography (OCT) produces cross-sectional images RNFL of the retina allowing measurement of RNFL thickness.2 Polar- monkeys by enhanced polarization-sensitive optical coherence ization-sensitive OCT (PS-OCT)3 and scanning laser polarimetry tomography (EPS-OCT). The monkeys were perfused with glu- (GDx; Carl Zeiss Meditec, Inc. Dublin, CA)4 measure phase taraldehyde and the eyes were enucleated and prepared for retardation in the RNFL that arises from anisotropic structures transmission electron microscopy (TEM) histologic analysis. in the RNFL.5 Changes in RNFL birefringence may precede Morphologic measurements from TEM images were used to ␳ RGC death. estimate neurotubule density ( RNFL), axoplasmic area (Ax) Birefringence (⌬n) is a dimensionless measure of the anisot- mode, axon area (Aa) mode, slope (u) of the number of neu- ropy of the refractive index (n) of a material and is given by the rotubules versus axoplasmic area (neurotubule packing den- difference between the extraordinary index (n ) and ordinary sity), fractional area of axoplasm in the nerve fiber bundle (f), e index (no), where no and ne are the refractive indices for mitochondrial fractional area in the nerve fiber bundle (xm), polarization perpendicular and parallel to the axis of anisot- mitochondria-containing axon profile fraction (mp), and length ⌬ ϭ Ϫ ropy respectively ( n ne no). Phase retardation is propor- of axonal membrane profiles per unit of nerve fiber bundle tional to birefringence according to the expression ⌬n ϭ area (L /A ). Registered PR/UD and morphologic parameters ␭ ⌬ ␭ am b 0PR/360° Z where 0 is the free-space wavelength, PR is the from corresponding angular sections were then correlated by phase retardation, and ⌬Z is the thickness of the material.6 In using Pearson’s correlation and multilevel models. general, ⌬n of the RNFL is weakly wavelength dependent 7 RESULTS. In one eye there was a statistically significant correla- across visible and near infrared wavelengths. The RNFL dem- ␳ ϭ ϭ tion between PR/UD and RNFL (r 0.67, P 0.005) and onstrates form birefringence that originates from anisotropic between PR/UD and neurotubule packing density (r ϭ 0.70, cylindrically shaped cellular structures in the RGC axons.8,9 P ϭ 0.002). Correlation coefficients of r ϭ 0.81 (P ϭ 0.01) and The major cylindrical structures of the RGC axonal cytoskele- r ϭ 0.50 (P ϭ 0.05) were observed between the PR/UD and A ton are neurotubules (NT), neurofilaments, and neurotubule- x 10 5 modes for each respective subject. associated proteins. Huang and Knighton identify neurotubules as the source of birefringence from the RNFL. CONCLUSIONS. Neurotubules are the primary source of birefrin- Theoretical analysis has attributed RNFL reflectance to the gence in the RNFL of the primate retina. (Invest Ophthalmol cylindrically shaped mitochondria and axonal membranes.9 Vis Sci. 2009;50:5238–5246) DOI:10.1167/iovs.08-3263 It is postulated that either changes in neurotubule density of RNFL bundles or neurotubule packing densities within the axons themselves are responsible for the differences in bire- 11 1 ␳ From the Biomedical Engineering Laser Laboratories and the fringence surrounding the ONH. Neurotubule density ( RNFL) 4Biomedical Informatics Lab, Department of Biomedical Engineering, is defined in this report as a scalar estimate of the number of The University of Texas at Austin, Austin, Texas; the 2U.S. Air Force neurotubules per unit area of RNFL tissue (NT/␮m2). There is 3 ␳ Research Laboratory, Brooks City-Base, Texas; and the Department of strong evidence that the regional characteristic RNFL surround- Neuropathology and Ophthalmic Pathology at the Armed Forces Insti- ing the optic nerve head (ONH) is a source of birefrin- tute of Pathology (AFIP), Washington, DC. 5,12,13 ␳ gence, but it has not been clear whether RNFL is the only Supported by Grant R01EY016462-01A1 from the National Eye source of birefringence.9 RNFL birefringence has been investi- Institute at the National Institutes of Health and Long Term Full Time Program Grant BV0700313000. Any opinions, interpretations, conclu- gated as a possible diagnostic to detect early subcellular sions, and recommendations are not necessarily endorsed by the U.S. changes in glaucoma before there is any measurable change in 5,12,13 13 Air Force. RNFL thickness. Fortune et al. injected colchicine into Submitted for publication December 4, 2008; revised April 7, the vitreous of nonhuman primate eyes and observed neuro- 2009; accepted July 30, 2009. tubule disruption with a reduction of RNFL birefringence with- Disclosure: G.M. Pocock, None; R.G. Aranibar, None; N.J. out any accompanying change in RNFL thickness. These results Kemp, None; C.S. Specht, None; M.K. Markey, None; H.G. Ry- show that decreases in the number of neurotubules occur lander III, None ␳ before thinning of the RNFL, and in situ measurement of RNFL The publication costs of this article were defrayed in part by page can be used as a diagnostic for cytoskeletal changes in RGC charge payment. This article must therefore be marked “advertise- 13 ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. axons. In this study, we compared the measured birefrin- Corresponding author: H. Grady Rylander III, The University of gence signal in the peripapillary retina with the corresponding Texas at Austin, 1 University Station, C0800; Austin, TX 78712; measured anatomic features to explore the origin of the RNFL [email protected]. birefringence.

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METHODS Measurement of RNFL Birefringence The retinas of two healthy 6-year-old female, 5.9 and 7.7 kg, cynomolgus monkeys (subjects 1853 and 57204) were imaged over a period of 62 days. Intraocular pressures and subjective visual function were normal. All experimental procedures were approved by The University of Texas at Austin Institutional Animal Care and Use Committee (IACUC, Protocol 05021401) and conformed to all United States De- partment of Agriculture (USDA), National Institutes of Health (NIH) guidelines, and the ARVO Statement for the Use of Animals in Oph- thalmic and Vision Research. A combination of IM ketamine (10 mg/ kg) and xylazine (0.25 mg/kg) was used to anesthetize the monkeys, and anesthesia levels were monitored by a certiﬁed veterinary technol- ogist. A retrobulbar injection of 0.5% xylocaine and 0.375% marcaine was given to immobilize the eye that was imaged. Pupils were dilated by using 1 drop of 1% cyclopentolate and 1 drop of 1% tropicamide. One drop of 10% methylcellulose, to prevent dehydration, was placed in the eye to be imaged, and a contact lens was placed on that eye. A contact lens that rendered the monkey slightly myopic was used to ensure that light was focused on the internal limiting membrane (ILM).

The peripapillary RNFL thickness (ZRNFL) 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 ⌬n with high sensitivity in weakly birefringent ␭ ϭ tissues. The system utilizes a mode-locked Ti:Al2O3 laser source ( 0 ⌬␭ ϭ 830 nm, 55 nm) linearly polarized at 45°. The ZRNFL 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 ␮m of RNFL ␮ thickness (degrees/per 100 m) by dividing local PR by ZRNFL. The PR map was measured in an area and ring scan configuration. Each area map consisted of 24 evenly spaced (15°) radial scans containing 2, 6, or 8 clusters distributed uniformly between 1.4 and 1.9 mm from the center of the optic nerve head. A cluster comprised 36 or 64 A-scans individually acquired over a respective 0.9- or 1.6-mm2 area. Ring scans are derived from sector scans spread 5° apart. The right eye FIGURE 1. The peripapillary RNFL was divided into 8 and 16 angular of each subject was imaged twice on separate days, to assess repro- sections. Each angular section is labeled by the symbols superimposed ducibility of retinal birefringence measurements. on the area surrounding the ONH (white circle) in subjects (A) 1853 A single peripapillary map was acquired in approximately 45 min- OS and (B) 57204 OD, The uncertainty in RNFL bundle positions are Ϯ Ϯ utes. Laser power incident on the cornea was 2.8 mW during lateral 22.5° and 11.25° for subjects 1853 and 57204, respectively. Adapted with permission from Hogan MJ, Alvarado JA, Weddell JE. scanning and 1.7 mW while stationary for both scan configurations. Histology of the Human Eye. Philadelphia: WB Saunders; 1971. Approximate laser spot size at the retinal surface was 30 ␮m. Axial resolution was 5 ␮m (determined by the coherence length of the laser source in air). 7.4) consisting of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer. Each animal was first brought down to a RGC Axon Organelle Sampling surgical level of anesthesia with a combination of IM ketamine (10 The PR/UD sinusoid pattern11,15,16 surrounding the ONH was used to mg/kg) and xylazine (0.5 mg/kg). Pentobarbital sodium (10 mg/kg) determine the angular interval around the ONH that RGC axon or- was given first for surgical anesthesia and then an IV overdose was ganelles must be sampled. Using the Nyquist criterion, RGC axon administered. organelles were measured in eight angular sections (octants) of the After each perfusion was complete, all four eyes were enucleated peripapillary retina in the first eye sampled (1853 OS). Based on the and immersed in 180 mL of the primary fixative solution from 1 to 3 ␳ correlation result between birefringence and RNFL of the first eye, it hours before dissection from the periorbital tissue and removal of the was determined that 16 angular interval sections should be sampled posterior hemisphere of the eye. A 360° incision was made at the ora around the ONH of the second eye (decreasing bundle variance within serrata, and the anterior eye structures including the ciliary body, iris, a region) to increase the power to 80% and detect a significant corre- lens, and cornea were removed from the posterior eye cup. A notch ␳ lation between RNFL and birefringence (Fig. 1). Each bundle within an and suture placed in the nasal sclera before enucleation remained angular section was chosen at random to eliminate bias. Nerve bundles visible throughout the specimen preparation process to maintain were sampled from a 0.15-mm2 rectangular area from each angular proper orientation. All posterior eye cups were then simultaneously section, which contained approximately 15 to 20 nerve fiber bundles; immersed in 180 mL of the primary fixative solution for approximately therefore, 3 nerve fiber bundles approximate 15% to 20% and 7.5% to 1.5 hours. 10% of the sampled area of subjects 57204 and 1853, respectively. The Eye cups were washed in three 30-minute changes of 0.1 M sodium exact number of bundles within each angular section was not known. phosphate buffer (180 mL each, pH 7.4) and postfixed in 90 mL of 2% osmium tetroxide (aqueous) solution for 1 hour. They were washed Preparation of Ocular Tissue again two times for 30 minutes per wash in 0.1 M sodium phosphate Two days after EPS-OCT imaging was completed, both monkeys were buffer (180 mL each, pH 7.4) and then one more time in 180 mL of anesthetized and transcardially perfused with a fixative solution (pH distilled water for 30 minutes. The eye cups were then dehydrated

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through a series of increasing ethanol concentration solutions (50% for 14 hours, 75% for 1 hour, 100% for 1 hour, and 100% for 1 hour, all in 180 mL) followed by two immersions in acetone (50 mL 100% acetone for 1 hour each). The posterior eye cups were then infused with 1:3 low-viscosity resin (EMBed-812; Electron Microscopy Sciences, Hat- field, PA) in acetone for 1 hour, after which they were covered and left in 2:3 resin for 19 hours overnight. The following day, the eye cups were placed into fresh solutions of 100% resin and left in uncovered containers for approximately 5 hours. They were then placed anterior side down into cubical embedding molds. The molds were put into a 60°C oven for 21 hours overnight, during which the resin solution polymerized, and the eye cups were removed the following day. Superficial lines intersecting at the center of the ONH profile were used to divide the cube mold of the embedded eye into wedge-shaped angular sections. Each sample wedge was carefully carved from the cube to prevent excess tissue removal surrounding the ONH. Glass knives were used to create a flat rectangular block face (500 ϫ 300 ␮m). Transmission Electron Microscopy Ultrathin and semithin sections were sampled 1.6 to 1.9 mm from the ONH in regions (Ultracut UCT Ultramicrotome; Leica Microsystems GmbH, Ernst-Leitz-Strasse, Germany). Semithin sections (0.5 ␮m) were cut and stained with toluidine blue before ultrathin sections (60 nm) to ensure that the block’s orientation on the microtome would result in transverse cuts. Several ultrathin sections were placed on copper, ␣-numeric–indexed grids (Electron Microscopy Sciences) and stained FIGURE 2. Low-magnification (2200ϫ) image of a RNFL bundle from ␮ with 2% uranium acetate and 0.3% lead citrate before TEM analysis. subject 57204 in angular section NSI. Scale bar, 2 m. Gray-scale digital images were captured on a transmission electron microscope (EM208 TEM; Phillips, Eindhoven, The Netherlands)

equipped with a digital camera system (Advantage HR 1 MB; AMT, the total organelle area (Am and Ao) for each RGC axon and was then Danvers, MA). Each bundle was photographed at low (2,200ϫ– plotted against the respective number of neurotubules (k) in a scatter

2,800ϫ), medium (11,000ϫ–14,000ϫ), and high (28,000ϫ–44,000ϫ) plot (Excel; Microsoft, Redmond, WA) to determine k(Ax). Axoplasmic magnification. area probability distribution functions (p[Ax]) were then computed for each bundle using a kernel estimation method based on an Epanech- Identification of RGC Axons and Organelles nikov kernel function. A Freedman-Diaconis bin width rule was used to determine the bandwidth of the kernel smoothing window and was All morphologic measurements were made using Image J (ver. 1.36; adjusted using Scott’s skewness factor. The axoplasmic fractional area developed by Wayne Rasband, National Institutes of Health, Bethesda, in the bundle (f ) is determined by summing all A measurements in MD; available at http://rsb.info.nih.gov/ij/index.html) on a laptop com- x x the bundle and then dividing the result by the area of the bundle (A ): puter (Hewlett Packard Compaq, Palo Alto, CA). Low magnification b

TEM images were used to measure nerve fiber bundle area (Ab; Fig. 2). A montage of each nerve fiber bundle was created from a series of ͸N medium-magnification images (Photoshop, ver. 8.0; Adobe Systems, Ax,i ϭ San Jose, CA; Fig. 3). RGC axons were captured in high-magnification ϭ i 1 fx (1) images to measure respective axonal cross-sectional area (Aa), neuro- Ab tubule number (k), mitochondrial area (Am), noncytoskeletal organelle areas (A ), and unidentifiable features of the RNFL bundle. Neurotu- o Calculations of k(Ax), p(Ax), and fx are used to estimate RNFL neuro- bules in each of the selected RGC axons were manually counted using tubule density in the nonsparse axons of the nerve fiber bundle the Cell Counter plug-in feature in Image J. A single person identified according to the expression: and counted the RGC organelles in subject 1853 and two different people identified and counted organelles for subject 57204. ͑ ͒ k Ax The RNFL anterior and posterior boundaries were defined as the ␳ ϭ f ͵ p͑A ͒ dA (2) RNFL x x x ILM vitreous interface and the anterior-most RGC bodies,17 respec- Ax

tively. If RGC bodies were not present directly posterior to the nerve Ax fiber bundle, then the posterior boundary was defined at the points ␳ were RGC axons were absent. A line that bisected Mu¨ller cell processes where RNFL is given in units of number of neurotubules per unit RNFL and their footplates at the ILM defined boundaries between adjacent area. nerve fiber bundles. Axons that appeared pale and had substantially The right side of equation 2 is essentially a twice-weighted sum of fewer neurotubules were identified as “sparse” axons and were re- RGC axoplasmic neurotubule densities. corded apart from the “nonsparse” axons (Fig. 4). Amacrine cells or ͑ ͒ glial cells were not included in RGC axons counts when criteria k Ax 18 ␳ ͑ ͒ ϭ described previously were used. x Ax (3) Ax Neurotubule Density Estimation which are estimated by the k(Ax) linear regression model. Individual ␳ Nonsparse Axons. RGC axoplasmic area (Ax) measurements estimates of neurotubule counts for each axon ( x) are weighted by the measured from TEM images were used in a statistical algorithm to probability of a given Ax in the nerve fiber bundle (p[Ax]dAx) and by ␳ estimate RNFL.(Ax) was calculated as the difference between (Aa) and the fraction of axoplasmic areas in the bundle (fx) which accounts for

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FIGURE 3. (A) A montage of a nerve ﬁber bundle from subject

57204 in angular section ITL. Mon- tages were transferred to a tablet PC, where cellular structures were traced, counted, and measured. The montage was used to keep track of photographed high-magnification images of RGC axons using the Image J’s Region of Interest (ROI) Manager to ensure that there were not any repeat or missed images of any RGC axons within the nerve fiber bundle. (B) Medium magnification (11,000ϫ–14,000ϫ) TEM image used to create the montage in (A). Scale bar, (A)2␮m; (B) 1 ␮m.

␳ null RNFL values in non-RGC areas of the RNFL bundle and effectively to compute the correlation between them (Fig. 5). The shift was used ␳ ␳ ␳ normalizes x into RNFL. to avoid PR/UD and RNFL measurement positions taken at angular Sparse Axons. A different statistical method is used to estimate boundaries between sections in both subjects. ␳ RNFL in the temporal maculopapillary fibers (angular sections TI,TSI, Two approaches were used to find a correlation between PR/UD and TIS) for both subjects. The Ax range for the nonsparse axons in this and RGC organelles. The first approach was a Pearson product moment region was a narrow range and resulted in a low R2 in linear fits of k correlation. Correlation coefficients and significance values were com-

versus Ax. puted (SAS ver. 9; SAS, Cary, NC). One hundred nonsparse axons were selected from the angular A multilevel modeling technique was used for the second ap- regions. The morphologic characteristics of the selected axons resem- proach, since it allows PR/UD and RGC organelles to be correlated on bled nonsparse axons in other regions of the eye. The sample set was two levels so that ﬁner angular resolution of PR/UD measurements used to generate an average nonsparse axoplasmic neurotubule density could be preserved. Multilevel modeling was implemented through the ␳ ( nsx) from which a 25% neurotubule density threshold was set to procedure PROC MIXED within the statistical analysis software. The classify sparse axons in the respective region. Once sparse axons were model accounts for the random effect of angular position of PR/UD ␳ identiﬁed, a sparse axon neurotubule density ( sx) was determined. measurements. ␳ ␳ The nsx and sx were individually weighted by their respective fx and ␳ summed to get an estimate for RNFL in the respective section according to: RESULTS

␳ ϭ ⅐ ␳៮ ϩ ⅐ ␳៮ Primate RNFL Birefringence RNFL fx,ns x,ns fx,s x,s (4) Experimental Correlation between Birefringence Spatially resolved RNFL peripapillary PR/UD maps were ob- tained by EPS-OCT from four eyes of two subjects. Results from and RGC Organelles both subjects are presented throughout the results section to Radial area and ring scan PR/UD measurements falling within an demonstrate the similarities and differences in birefringence angular section were averaged, resulting in an equal number of PR/UD and morphologic measurements. ␳ values for every mean RNFL. The averaged PR/UD measurements for Radial area and ring scan measurements of PR/UD were subjects 1853 and 57204 resulted in Ϯ22.5° and Ϯ11.25° of angular relatively higher in the superior and inferior areas in both error, respectively. Average PR/UD measurement positions were opti- subjects (Fig. 6). Clusters of PR/UD radial area and ring scans ␳ mally registered with angular RGC axon organelle mean measurements within respective RNFL angular section boundaries were aver-

FIGURE 4. High-magnification images (28,000ϫ–44,000ϫ) were used to measure RGC axon neurotubules and organelles. Sparse axons were rare in nerve fiber bundles except in the temporal macular region of the eye in both subjects. Axons that had a neurotubule density that was less than 25% percent of the average neurotubule density of the axon population were classified as sparse axons. (A) Sparse axons (S) appeared pale and had fewer neurotubules. (B)An RGC axon from nasal region of subject 57204. Scale bar, 0.5 ␮m.

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Primate RNFL Neurotubule Density ␳ Estimates of RNFL for all bundles in both eyes are given in Tables 2 and 3 and are shown graphically in Figure 7A. Neu- rotubule 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 RNFL between the two subjects (Fig. 7B). The result was a correlation coefficient of 0.97 (P Ͻ 0.001). ␳ The linear relationship between k and Ax used in the 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 investi- gating axon size and neurotubule density in the optic nerve head.19 Correlation between Birefringence and RGC Organelles Subject 57204 had a correlation coefficient of 0.67 (P ϭ 0.005) ␳ between PR/UD and RNFL (Table 4), and the multilevel model resulted in standardized regression coefficients of 0.50 (P ϭ 0.04). The averaged pattern of PR/UD is shown superimposed ␳ on respective RNFL for both subjects in Figure 9.

␳ FIGURE 5. (A) Original positions of the mean RNFL and PR/UD radial ␳ area and ring scan measurements used for correlation with RNFL for subject 57204. Yellow and blue bars: the original position of angular

section TIS and the lowest PR/UD measurements within an angular section, respectively. (B) Radial area and ring scans of PR/UD falling within an angular section were averaged, resulting in an equal number ␳ of PR/UD measurements for every RNFL. Green bar: the optimally ␳ registered RNFL for angular section TIS and PR/UD measurement. N, nasal; T, temporal; S, superior; I, inferior.

aged, resulting in an equal number of PR/UD measurements for ␳ every RNFL value. The 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 Ͻ 0.05) for subject 1853. All quadrant pairs excluding the inferior and nasal pair were signiﬁcantly different (P Ͻ 0.05) for subject 57204. 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 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 FIGURE 6. PR/UD (proportional to birefringence) measurements square root of the number of clusters. The highest SE of all along a 360° circular sweep around the ONH. PR/UD measurements PR/UD measurements was Ϯ7.1 deg/100 ␮m in the nasal (ring or radial) in each eye are plotted with a unique symbol. Black region. The average SE of all three scans was Ϯ1.9 deg/100 ␮m squares: the average PR/UD of combined ring and radial scans for each angular section region that was used to determine neurotubule density. ⌬ ϭ ϫ Ϫ4 ( n 0.4 10 ) and was calculated by dividing the average (A) Two different radial area and single ring scan for subject 57204. (B) SD of cluster PR/UD measurements from all three scans by the Area scan and ring scan of 1853 OS. N, nasal; T, temporal; S, superior; square root of the number of scans. I, inferior.

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TABLE 1. PR/UD (Proportional to Birefringence) Quadrant Means of Radial Area Scan and Ring Scan Measurements in One Eye of Each Subject

57204 OD 1853 OS

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

Scan Pattern 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

Morphologic measurements from TEM images were used to 57204 and may have resulted from a thinner RNFL area or eye 15 estimate mean values of axoplasmic area (Ax) mode, axon area movements. This finding was similar to others. (Aa) mode, slope (u) of neurotubule number versus axoplasmic Uncertainty in the PR/UD measurements comes from any area (neurotubule packing density), fractional area of axoplasm error in the RNFL thickness (ZRFNL) measured from EPS-OCT in the nerve fiber bundle (fx), mitochondrial fractional area in intensity images and phase retardation (PR) estimates from the the nerve fiber bundle (xm), mitochondria-containing axon EPS-OCT algorithm. Error in the ZRFNL comes from the refrac- ϭ 20 profile fraction (mp), and length of axonal membrane profiles tive index range for the RNFL (4%, n 1.34–1.39) and per unit nerve fiber bundle (Lam/Ab). uncertainty in the exact location of the RNFL boundaries due Pearson product moment correlation coefficients of the to the axial resolution limit of the EPS-OCT instrument (5 ␮m). morphologic parameters with PR/UD are summarized in Table Noise creates an uncertainty of approximately Ϯ1° for PR 5. Correlation coefficients of r ϭ 0.81 (P ϭ 0.01) and r ϭ 0.5 estimates. Thus, uncertainty in PR is fractionally greater in ϭ (P 0.05) were observed between PR/UD and mean Ax mode regions with low PR such as the nasal and temporal regions. in subjects 1853 and 57204 (Fig. 10), respectively. Mean Ax Radial and rotational error in position of the eye during imag- mode values were similar in the superior and inferior portions ing may have caused a registration error in PR/UD measure- of both eyes and were larger than temporal and nasal portions. ments.

Mean Aa and PR/UD yielded a significant correlation coefficient of r ϭ 0.79 (P ϭ 0.01) in subject 1853 (Fig. 10B). Neurotubule Density of the Primate RNFL ϭ In subject 57204, a significant correlation coefficient of r Sources of uncertainty for ␳ determination include: sam- ϭ RNFL 0.7 (P 0.002) was observed between section averaged pling RNFL bundles near the angular section boundaries, mis- PR/UD and the k versus Ax regression fit slope (u) that is used classified cells or cellular structures, different observers for the as a measure of neurotubule packing density (Fig. 11). Arcuate two eyes studied, and sampling location from the ONH for bundle regions had the highest packing density, followed by ␳ each eye. The higher RNFL values for subject 1853 can be the nasal region, and last the papillomacular region. explained by counter bias between the first and second eye. The mean difference (95% limits of agreement) between neu- DISCUSSION rotubule counts of the same RGC axons of two counters for subject 57204 was Ϫ1.6(Ϫ8.8 to 5.6) neurotubules. ␳ Primate RNFL Birefringence Some angular sections had more variability in RNFL within PR/UD measurements were highest superior and inferior to nerve bundles than others. Most of the variability could be attributed to differences in RNFL nerve bundle area (Ab) mea- the ONH for both subjects. In subject 57204, the average of the ␳ quadrant PR/UD values given in Table 1 for the superior and surements, since RNFL estimates are calculated using Ab. The Ϯ ␮ ⌬ ϭ ϫ Ϫ4 standard deviation of RNFL thickness of human retina ranges temporal region are 18.2 1.0 deg/100 m( n 4.2 10 ) ␮ ␮ and 9.3 Ϯ 0.3 deg/100 ␮m(⌬n ϭ 2.1 ϫ 10Ϫ4). Cense et al.15 from 15 m in the temporal region to 26.5 m in the inferior measured the RNFL birefringence of two human subjects in vivo using PS-OCT at 840 nm. They also reported higher RNFL TABLE 3. Estimated Neurotubule Densities in Three Nerve Fiber Ϫ4 birefringence (4.1 ϫ 10 ) in the superior and inferior areas Bundles of Each RNFL Section with the lowest in the temporal region (1.2 ϫ 10Ϫ4). Huang et al.16 calculated similar results of averaged birefringence for the Section Bundle 1 Bundle 2 Bundle 3 Mean SD superior (4.2 ϫ 10Ϫ4) and temporal region (2.5 ϫ 10Ϫ4)of12 human eyes. The SE was greatest in the nasal region for subject NSI 53.7 42.6 37.1 44.5 8.5 NSS 66.9 54.2 36.1 52.4 15.5 SNL 77.0 45.1 68.4 63.5 16.5 TABLE 2. Estimated Neurotubule Densities in Three Nerve Fiber SNM 87.7 66.9 68.3 74.3 11.6 Bundles of Each RNFL Octant STM 45.1 59.8 45.8 50.2 8.3 STL 63.0 18.1 60.8 47.3 25.3 Section Bundle 1 Bundle 2 Bundle 3 Mean SD TSS 33.0 26.2 33.5 30.9 4.1 TSI 4.5 9.3 33.1 15.6 15.3 NS 66.0 72.2 76.3 71.5 5.2 TIS 11.3 5.6 3.7 6.9 3.9 SN 110.1 69.8 64.6 81.5 24.9 TII 46.5 35.0 42.3 41.3 5.8 ST 126.5 79.4 85.4 97.1 25.7 ITL 72.6 24.5 83.5 60.2 31.4 TS 69.9 64.7 69.6 68.1 2.9 ITM 50.6 65.6 47.2 54.5 9.8 TI 32.5 28.8 37.6 33.0 4.4 INM 42.1 73.7 41.4 52.4 18.5 IT 80.5 96.3 69.9 82.2 13.3 INL 73.7 22.3 25.6 40.5 28.8 IN 98.4 77.0 68.3 81.2 15.5 NII 18.4 73.0 76.7 56.0 32.6 NI 49.6 74.6 90.1 71.4 20.4 NIS 17.7 42.0 38.8 32.9 13.2

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␳ FIGURE 7. (A) Mean RNFL estimates averaged over three nerve ﬁber FIGURE 8. (A) Axoplasmic area probability density function from an- bundles are presented for both subjects. (B) ␳ estimates for subject gular section NSS of 57204 OD used in the statistical algorithm to RNFL ␳ 57204 within angular section octants analogous to subject 1853 were estimate RNFL.(B) Least-squares linear ﬁts of neurotubule number (k) ␳ further averaged to produce eight RNFL values for comparison with versus axoplasmic area (Ax). those in subject 1853. N, nasal; T, temporal; S, superior; I, inferior.

21 ␳ Ϯ region. Standard deviations of RNFL greater than 20 NT/ the pattern was not unique to a single eye; however, it is ␮m2 occurred in the arcuate bundles and the bundles located possible that the neurotubules in the small axons of the pap- in the inferior nasal portion of the RNFL near major blood illomacular bundle degenerated faster than the large axons vessels. Bundles lying close or adjacent to temporal blood found elsewhere. The neurotubules polymerize and depoly- vessels that approximately overlie the arcuate nerve bundles22 merize very quickly and the smaller axons may be more fragile were not excluded from the sampled population. The few than the larger ones. The sparse axons and fewer nonsparse 18,22–24 histologic studies of primate RNFL thickness did not axons within octant TI and angular sections TSI and TIS could measure thickness near large blood vessels because RNFL bor- also be attributed to bilateral optic atrophy (BOA).28 25 ders could not be accurately determined. The RNFL bundle The packing density was greatest for sections SNL,SNM,STM, area variation near and away from blood vessels as well judg- ITM, and INM which are sampled from arcuate ﬁbers. The ment as to the location of the RNFL borders would produce arcuate portions of the RNFL are frequently involved in glau- ␳ 27 variability in RNFL estimates. coma and optic neuropathies. Higher birefringence in the Glial content (Mu¨ller cells and astrocytes) of nerve bundles superior and inferior region of the eye can be attributed to a would also contribute to bundle area variation seen within the combination of higher neurotubule packing density, larger

same angular section since glial tissue is included in the Ab axons, and less compartmentalization by glial tissue than other measurement. Ogden22 reported that the proportion of RNFL regions of the eye.27 bundle area occupied by glia is independent of RNFL thickness and does not vary regularly with distance from the ONH in Correlation between Birefringence and RGC cynomolgus and rhesus monkeys. Angular sections with the Axon Organelles ␳ highest variability in RNFL estimates also had the largest variability in glial content percentage. The high correlation between Ax and PR/UD in both eyes is

Sections TSI,TIS, and TI are sampled from the papillomacu- interesting because axoplasmic regions of RGC axons are the only lar bundle fibers and have the smallest RGC fibers.26A low ␳ mean RNFL may be attributable to a combination of a thin 23 27 ␳ RNFL and low number of RGC axons that have a signifi- TABLE 4. Correlation between PR/UD and RNFL in Subject 57204 cantly smaller mean neurotubule number. The sparse axons in the papillomacular bundles could be a fixation artifact if the Statistical Model rP smaller axons were not adequately preserved by the perfusion Pearson’s correlation 0.67 0.005 of fixative. A similar pattern of sparse axons was observed in Multilevel model 0.5 0.04 the two eyes studied which should give some assurance that

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FIGURE 9. Average birefringence measurements calculated from EPS- FIGURE 10. PR/UD (proportional to birefringence) measurements OCT PR/UD radial area and ring scans plotted with respective regional from radial area and ring scans in subject 57204 were averaged within ␳ RNFL for subjects (A) 57204 (B) and 1853. Area and ring scans of PR/UD its respective angular section around the (ONH) and superimposed on measured within angular regions that were sampled for neurotubule (A) mean (Ax) modes for subject 57204 and (B) mean (Aa) and (Ax) density surrounding the ONH were averaged within the respective angu- modes for subject 1853 measured in corresponding regions of the lar section region, resulting in an equal number of PR/UD measurements same eye. N, nasal; T, temporal; S, superior; I, inferior. ␳ for every RNFL value. N, nasal; T, temporal; S, superior; I, inferior. source of the birefringence signal. The mean (u) (neurotubule areas that neurotubules can inhabit. In addition, there was a packing density) was more significantly correlated with PR/UD ␳ significant correlation between Aa and PR/UD in subject 1853. than the mean RNFL for subject 57204. This suggests that the These findings suggest that a structure within the axoplasm is the birefringence surrounding the ONH results from a difference in

TABLE 5. RGC Morphologic measurements Correlated with Phase Retardation Per Unit Depth (PR/UD, Proportional to Birefringence) for Subjects 57205 and 1853

57204 OD 1853 OS

RGC Axon Morphologic Measurement Abbreviation rPrP

Least-squares linear slope (number of NTs per unit Ax area), NT/␮m2 Mean u 0.70* 0.0028 0.68 0.0941 ␮ 2 Mean RGC axoplasmic area mode, m Mean Ax 0.50* 0.0500 0.81* 0.0145 ␮ 2 Mean axon area mode, m Mean Aa 0.47 0.0600 0.79* 0.0184 Neurotubule density (number of NTs per unit RNFL area), ␮ 2 ␳ NT/ m Mean 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 xm 0.02 0.9500 — — Mitochondria-containing axon proﬁle fraction (per unit RNFL

bundle area), % Mean mp — — 0.24 0.5708 ␮ Ϫ1 Axonal membrane length (per unit RNFL bundle area), m Mean Lam/Ab 0.24 0.3670 0.23 0.5811

RGC axoplasmic area (Ax) mode, neurotubule (NT) number (k) versus Ax least-squares linear slope (u), and fractional area of axoplasm per unit nerve fiber bundle (fx) measurements are associated with nonsparse axons only. Axon area (Aa) mode, mitochondrial fractional area (xm), mitochondria-containing axon profile fraction (mp), and length of axonal membrane per unit nerve fiber bundle area (Lam/Ab) measurements are associated with both sparse and nonsparse axons. * Morphologic parameters that had a significant correlation with PR/UD.

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6. Bass M, ed. Handbook of Optics. Classical Optics, Vision Optics, X-Ray Optics. Vol 3. 2nd ed. New York: McGraw-Hill Professional; 2000. 7. Huang XR, Knighton RW. Linear birefringence of the retinal nerve fiber layer measured in vitro with a mulitspectal imaging micropo- larimeter. J Biomed Opt. 2002;7:199–204. 8. Huang X-R, Knighton RW. Theoretical model of the polarization properties of the retinal nerve fiber layer in reflection. Appl Opt. 2003;42:5726–5736. 9. Zhou Q, Knighton RW. Light scattering and form birefringence of parallel cylindrical arrays that represent cellular organelles of the retinal nerve fiber layer. Appl Opt. 1997;36:2273–2285. 10. Balaratnasinga C, Morgan WH, Bass L, Matich G, Cringle SJ, Yu D. Axonal transport and cytoskeletal changes in the laminar regions after elevated intraocular pressure. Invest Opthalmol Vis Sci. 2007;48:3632–3644. 11. Rylander HG, Kemp NJ, Park JS, Zaatari HN, Milner TE. Birefrin- gence of the primate retinal nerve fiber layer. Exp Eye Res. 2005; FIGURE 11. PR/UD (proportional to birefringence) measurements from 81:81–89. radial area and ring scans for subject 57204 were averaged within its 12. Fortune B, Cull GA, Burgoyen CF. Relative course of retinal nerve respective angular section around the ONH and superimposed on mean fiber layer birefringence and thickness and retinal function (u; neurotubule packing density) data measured in corresponding regions changes after optic nerve transection. Invest Ophthalmol Vis Sci. of the same eye. N, nasal; T, temporal; S, superior; I, inferior. 2008;49:4444–4452. 13. Fortune B, Wang L, Cull G, Cioffi GA. Intravitreal colchicine causes decreased RNFL birefringence without altering RNFL thickness. NT packing density of the axons themselves and not from a Invest Opthalmol Vis Sci. 2008;49:255–261. difference in the packing density of axons within bundles. 14. Kemp NJ, Park J, Zaatari HN, Rylander HG, Milner TE. High- Lam/Ab failed to correlate with PR/UD in subject 57204, sensitivity determination of birefringence in turbid media with despite increased sampling and further supports the finding29 enhanced polarization-sensitive optical coherence tomography. J that cell membranes are not the source of birefringence. Mito- Opt Soc Am A Opt Image Sci Vis. 2005;22:552–560. 15. Cense B, Chen TC, Park BH, Pierce MC, Boer JF. Thickness and chondrial fractional area (xm) and the percentage of mito- birefringence of healthy retinal nerve fiber layer tissue measured chondria-containing axon profiles (mp) did not correlate with PR/UD for subject 57204 and 1853, respectively. with polarization-sensitive optical coherence tomography. Invest Opthalmol Vis Sci. 2004;45:2606–2612. 16. Huang X-R, Bagga H, Greenfield DS, Knighton RW. Variation of CONCLUSION Peripapillary retinal nerve fiber layer birefringence in normal human subjects. Invest Opthalmol Vis Sci. 2004;45:3073–3080. Our findings further support the hypothesis that neurotubules 17. Quigley HA, Addicks EM. Quantitative studies of retinal nerve fiber are the primary source of birefringence in the RNFL. The layer defects. Arch Opthalmol. 1982;100:807–814. strongest correlation between ⌬n and PR/UD (P ϭ 0.0028) for 18. Perry VH. The ganglion cell layer of the mammalian retina. Prog all the morphologic parameters studied was the slope (u)of Retin Res. 1982;1:53–80. the regression fit of neurotubule number (k) versus axoplasmic 19. Hernandez C, Blackburn E, Alvarez J. Calibre and microtubule

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