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

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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 neurotu- bules 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. Investigative Ophthalmology & Visual Science, November 2009, Vol. 50, No. 11 5238 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/26/2021 IOVS, November 2009, Vol. 50, No. 11 Primate RNFL Birefringence and Neurotubules 5239 METHODS Measurement of RNFL Birefringence The retinas of two healthy 6-year-old female, 5.9 and 7.7 kg, cynomol- gus 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 tomogra- phy (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 con- taining 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.
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