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From the Devers Eye Institute, Legacy Health System, Portland, Oregon.
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Abstract |
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METHODS. Colchicine was injected into the vitreous cavity of one eye in each of six vervet monkeys (Chlorocebus sabaeus; estimated vitreal concentration: 1 mM, n = 3; 2 mM, n = 1; 10 mM, n = 2); an equivalent volume (approximately 0.1 mL) of sterile saline was injected into fellow control eyes. RNFL birefringence was measured by SLP before injection and every 10 minutes after injection for 2 hours. RNFL thickness was measured by OCT before injection and 2 hours later. After isolating each retina, biopsy specimens were obtained from the inferotemporal arcade region, approximately 2 mm from the center of the optic disc, using a 2-mm trephine and were processed for transmission electron microscopy (TEM). Retinas were then flat-mounted and stained with an antibody against polymerized β-III-tubulin.
RESULTS. RNFL birefringence measured by SLP decreased over time in all six colchicine-injected eyes, appearing to reach a plateau of –20% ± 7% (P < 0.0001) approximately 100 minutes after injection. There were no significant differences between quadrants (P = 0.44) and no apparent dose effect (P = 0.87). The change in vehicle-injected control eyes was –3% ± 3% (P = 0.06; NS). The change in RNFL thickness measured by OCT was +1% ± 4% (P = 0.81; NS) in colchicine-injected eyes and +6% ± 6% (P = 0.13; NS) in control eyes. There was no evidence of macular edema by fundus biomicroscopy, stereo fundus photography, or OCT. TEM revealed disorganization of microtubules, swelling of mitochondria, and blurred axonal membrane borders in colchicine-injected eyes. Flat-mounted retinas stained with an antibody against polymerized β-III-tubulin showed only a mild reduction of peripapillary stain intensity in the colchicine-injected eyes compared with controls.
CONCLUSIONS. Intravitreal injection of colchicine caused microtubule disruption within the axons of the RNFL in nonhuman primate eyes. This was manifest as a reduction of RNFL birefringence, without alteration of RNFL thickness, suggesting that such discrepancies can be informative about the status of axonal degeneration.
Yet ophthalmoscopic evaluation of the RNFL remains a challenging clinical task, especially in older patients with media opacities, miotic pupils, blonde fundi, or unsteady fixation. Photographic methods of RNFL assessment impose additional constraints because of the time delay and logistics of film development, and they are still greatly dependent on subjective judgments, which can vary between clinicians and across successive examinations.3 4 Moreover, even in the hands of experts, the minimum RNFL loss detectable by photography may be as great as approximately 50 to 70 µm.6 This has led to the development of objective digital imaging techniques for clinical assessment of the RNFL. Currently, the two techniques most capable of providing reproducible RNFL measurements are optical coherence tomography (OCT)7 8 9 and scanning laser polarimetry (SLP).10 11 The fundamentals of each technique, as well as their relative strengths, reproducibility, major sources of error, and limitations, have been carefully reviewed by others (see, for example, Stein et al.12 and Zangwill and Bowd13 ). Each technology is based on a different optical principle. OCT measures the relative delay in the time-of-flight of a near infra-red ("light") source after it is reflected by internal structures located at different retinal depths.14 OCT is thus able to provide a high-resolution cross-sectional image of the retina and to estimate the thickness of the RNFL by delineating the relatively steep reflectance transition at its anterior and posterior limits. In contrast, SLP estimates RNFL "thickness" by measuring the relative retardance of orthogonally polarized light. Retardance is caused by tissue property birefringence, which, in the case of the RNFL, is thought to depend on thin cylindrical parallel structures within the retinal ganglion cell (RGC) axons such as microtubules (MT) and neurofilaments (NF).15 16 17
Knighton et al.18 have speculated that morphologic changes within RGC axons, such as alterations of MT or NF, might precede irreversible axonal loss and should manifest as altered birefringence measurements (i.e., be measurable by SLP). If abnormalities of the birefringent elements within RGC axons were to develop before a decline in overall axonal caliber, then a temporal discrepancy between SLP and OCT measurements of the RNFL should occur, reflecting a potentially important pathologic state within RGC axons and a possible window for therapeutic intervention (i.e., a signal that RGCs might be "sick" but not yet dead and gone). The present study was thus designed as a proof of this principle: its purpose was to test the hypothesis that longitudinal differences between RNFL birefringence, measured by SLP, and RNFL thickness, measured by OCT, can exist and be informative about the state of axonal degeneration.
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Methods |
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Anesthesia
The experimental protocol began with the induction of general anesthesia with intramuscular injection of 15 mg/kg ketamine (Fort Dodge Animal Health, Fort Dodge, IA), along with a single subcutaneous injection of atropine sulfate (0.05 mg/kg). Animals were then intubated so that anesthesia could be maintained using 2% to 3% isoflurane (Baxter, Deerfield, IL) mixed with oxygen. Heart/pulse rate and arterial oxyhemoglobin saturation were monitored continuously (Propaq Encore model 206EL; Protocol Systems, Inc., Beaverton, OR) and were maintained above 80 min–1 and 95%, respectively. Body temperature was maintained with a warm-water heating pad set at 37°C. Pupils were fully dilated (
7 mm) with 1.0% tropicamide and 2.5% phenylephrine (Alcon Laboratories Inc., Fort Worth, TX). Topical anesthetic (0.5% proparacaine; Alcon Laboratories Inc.) and lubricating drops (Refresh; Allergan, Irvine, CA) were applied before insertion of a plano-powered, rigid gas-permeable contact lens in each eye.
Baseline Imaging
Before intravitreal injections, baseline images were obtained in each eye. Simultaneous stereoscopic photographs were taken of the optic disc and macula (3-Dx; Nidek Co., Ltd., Aichi, Japan), first without any filter, then with the camera’s red-free filter in place. SLP measurements were obtained using a GDxVCC instrument (Carl Zeiss Meditec, Inc., Jena, Germany). Anterior segment birefringence measurements were obtained before the initial baseline scan and were used to "compensate" all subsequent scans. Three SLP scans were averaged for the baseline and for each subsequent time point. The medium locus of RNFL values (the peripapillary profile or TSNIT curve) was exported for analysis. Each of the 64 values in the TSNIT series is an average from an 8-pixel-wide band centered on the optic disc.19 The outer limit of the medium band is 43 pixels from the center of the optic disc, so the center of the band has a radius of 39 pixels.19 This corresponds to a scan angle of 6.1°, which translates to a total diameter of 3.5 mm (assuming 45 µm per pixel for an emmetropic human eye with average axial length).19
OCT measurements of RNFL thickness were obtained using an OCT instrument (Stratus, v3.0.1; Carl Zeiss Meditec, Inc). The RNFL algorithm was applied with a 3.4-mm diameter scan centered on the optic disc. The 3.4-mm designation is more accurately stated as 3.46 mm for an emmetropic human eye with an axial length of 24.46 mm,20 which closely matches the center of the medium band (3.5-mm diameter) of the SLP. Empiric alignment procedures during pilot studies confirmed that the center of the medium band was a good match to the 3.4-mm diameter scan locus from the OCT in these relatively small vervet monkey eyes. Magnification differences resulting from the shorter axial length of the vervet eye would apply equally to both the SLP and the OCT instrument. The 3.46-mm diameter scan would have an actual transverse distance on the monkey retina of 2.9 mm (calculation based on measured axial length of 20 mm).20 Peripapillary retinal thickness (RT, using a 3.4-mm diameter scan) and macular thickness scans were also obtained in each eye during baseline and final time points.
Intravitreal Injections
Intravitreal injections were made through the temporal pars plana using a 30-gauge needle. Colchicine (Sigma-Aldrich, St. Louis, MO) was diluted into sterile balanced salt solution (Alcon Laboratories Inc.) to achieve an injected dose of 0.83 mg (in 0.083 mL), which provides an estimated vitreal concentration of 1 mM assuming a 2-mL vitreous volume (the latter based on measurements of vervet eye dimensions). This 1-mM dose was injected in three of the six experimental eyes; higher doses were injected in the remaining three experimental eyes (2 mM, n = 1; 10 mM, n = 2). The fellow (control) eye of each animal received an intravitreal injection of an equal volume (0.083–0.1 mL) of vehicle only (balanced salt solution).
Repeat Imaging
SLP scans were repeated in approximately 10-minute intervals; three scans were averaged at each of these time points. The series of postinjection time points consisted of three sequential time points in the experimental eye, followed by one time point in the control eye, which was then repeated twice more (see Results). After this series of SLP time points was completed, the postinjection OCT scans and fundus photographs were obtained. The duration of the entire experiment was approximately 2 hours. Note that one animal remained supine for 1 hour after receiving a 10-mM injection, reflecting an attempt to maximize and equalize colchicine concentration across the posterior pole. SLP imaging resumed at the 60-minute time point.
Histopathology
On completion of in vivo imaging, animals were immediately killed by intravenous injection (Euthasol; Diamond Animal Health, Inc., Des Moines, IA). After a bolus of IV heparin, perfusion fixation ensued through bilateral pre-cannulated carotid arteries using approximately 1 L 4% buffered paraformaldehyde. Eyes were enucleated, retinas were dissected, and the retinal pigment epithelium and vitreous were removed. With the use of a 2-mm trephine, a biopsy sample was obtained from the inferotemporal arcade region of each retina and prepared for transmission electron microscopy (TEM). A wedge-shaped incision was used to orient the sample so that ultrathin sections could be made perpendicular to the direction of RNFL bundles. Sections thus began tangentially along the biopsy edge closest to the optic disc.
After biopsy, each pair of retinas was stained simultaneously with mouse monoclonal antibodies against β-III-tubulin (TUJ-1 [Abcam Inc., Cambridge, MA] or SMI-62 [Covance Research Products Inc., Berkeley, CA]) according to the following protocol. Retinas were treated with 0.2% triton X-100 in 0.01 M PBS for 1 hour at room temperature, then with methanol and 0.3% H2O2 for 1 additional hour. Retinas were placed in blocking serum solution (2% horse serum and 2% BSA) for 12 hours, washed once with PBS, and incubated at 4°C with the primary antibody (1:500) for 5 days. They were then washed in 0.01 M PBS for 1 hour at least three times each. Finally, retinas were incubated at 4°C for 3 days with the FITC-conjugated horse anti–mouse secondary antibody (1:100) and were flat mounted for microscopy.
Statistical Analysis
Repeated-measures analysis of variance was used to test for the effects of treatment, time, and quadrant.
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Results |
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Figure 2 compares the group average (n = 6) TSNIT curves obtained at baseline (dashed curves) with those obtained at final time point (solid curves) for both the SLP measure of birefringence (Figs. 2A 2C) and the OCT measure of RNFL thickness (Figs. 2B 2D) . The data for colchicine-injected eyes are shown in the top row (Figs. 2A 2B) , and the data for control eyes are shown in the bottom row (Figs. 2C 2D) . The TSNIT average loss of 20% ± 7%, shown in Figure 1 , can be seen here again in Figure 2A for the SLP measure of RNFL birefringence. Also shown again is the lack of any significant change measured by SLP in control eyes (Fig. 2C) . Figures 2B and 2D show that there was no significant change in the OCT measure of RNFL thickness for either group; it increased by 1% ± 4% (P = 0.81, NS) in colchicine-injected eyes and increased by 6% ± 6% (P = 0.13, NS) in control eyes.
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Discussion |
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Evidence indicates that NFs become abnormal at an early stage of experimental glaucoma (specifically, an altered phosphorylation state)21 and that the largest axons expressing the most NFs are the most vulnerable in the same monkey model.22 It has also been shown that mRNA for NF decreases immediately after axotomy in the adult rat optic nerve, whereas β-tubulin mRNA initially increases.23 Perhaps even more relevant are the findings that a decrease in axonal caliber after optic nerve crush is delayed by approximately 1 month relative to the decrease in NF expression.24
It seems that more is known about changes in NF than MT after RGC injury, but the work of Knighton and colleagues15 16 17 suggests that MT are the primary source of birefringence in the RNFL. Although it is possible that other thin cylindrical parallel structures, such as NFs or the axonal membranes themselves, could also contribute to form birefringence of the RNFL,15 Knighton et al.17 found that RNFL birefringence declines to essentially zero after colchicine treatment of rat retinal explant preparations. The latter results are consistent with their earlier theoretical work15 and a model in which MT are the primary contributor to RNFL birefringence, with other mechanisms contributing less than 15%.17
The results obtained in the current experiment showed, on average, a 20% decline in the SLP-derived estimate of RNFL thickness around the optic nerve. The GDxVCC instrument measures retardance, then calculates its estimate of RNFL thickness using a linear conversion factor of 0.67 nm/µm10 (as stated in the instrument manual19 ). Because the conversion is linear, the decline in RNFL "thickness" measured by SLP represents a 20% decline in RNFL birefringence within approximately 100 minutes of intravitreal colchicine injection. This effect is smaller than that obtained by Huang and Knighton,17 which might be attributed to one or several differences in the experimental conditions. Their experiment was carried out on rat retinal explants with the use of a custom polarimeter device and a colchicine dose of 10 mM; our experiment was carried out in vivo using available clinical instrumentation and in doses ranging from 1 to 10 mM (it is difficult to achieve an estimated vitreous concentration of 10 mM because colchicine does not easily remain in solution at the concentration required before it is diluted into the vitreous volume). Although it is possible that the dose was not strong enough to observe the same magnitude of the effect obtained by Huang and Knighton17 (Huang X-R, personal communication, 2006), we did not see a substantive difference between the effects of 1 mM and 10 mM colchicine. Moreover, using intravitreal colchicine doses 10 to 100 times lower than what was used here, Davidson et al.25 found near total loss of neurotubules within the intraretinal axons of the monkey after only 1 hour.
In a separate study, Huang et al.26 also observed an approximately 50% decline in the reflectance of the rat RNFL after colchicine treatment. We did not see evidence of reflectance changes in the monkey RNFL, by clinical indirect ophthalmoscopy, inspection of fundus photography, or the strength of the internal reflectance signal on OCT. If proportional effects of colchicine on RNFL birefringence and reflectance followed that observed in the Huang et al.26 study, then reflectance should have declined by only approximately 10% in this study, which is perhaps below the detection threshold. It is also possible that some of the differences between our results and those of previous studies15 16 17 18 suggest that MT account for less than 85% to 100% of RNFL birefringence and less than 50% of RNFL reflectance in the primate eye.
There was a trend toward a greater effect of colchicine at the superior and inferior poles of the optic disc, where the RNFL is thickest (measured by OCT) and birefringence is greatest (measured by SLP), though our study was underpowered to characterize this relatively subtle differential spatial effect with statistical confidence. Variation in the degree of birefringence per unit thickness18 27 28 29 30 suggests that even in normal eyes not all bundles contain axons with the same density of birefringent elements. Consistent with those earlier findings, the ratio of birefringence to RNFL thickness was not uniform around the optic disc in our study, varying from approximately one third nasally and 30° temporal to the poles to a peak of two thirds or more at the superior and inferior poles. Even within a given bundle, not all axons contained an equivalent density of MT or NF. That was evident in all the TEM samples obtained in our study, and it is clearly demonstrated in Figures 9–71 through 9–75 in Hogan et al.31 This means that detecting birefringence changes relative to RNFL thickness will be complicated by normal variation not considered within the clinical instrument’s software, but it may also indicate that certain axons may be more or less susceptible to particular insults, depending on their internal structural composition. This could be an important avenue for future study in glaucoma or in other optic neuropathies or RGC diseases.
The observation that flat-mounted retinas stained with antibodies against β-III-tubulin did not consistently reveal differences between colchicine-injected and control eyes while the SLP measurement selectively showed reduced RNFL birefringence and the TEM results showed disorganized MT in colchicine-injected eyes suggests that the presence of MT subunits, even if more of less polymerized, is insufficient to produce normal RNFL birefringence. It seems that the three-dimensional MT architecture must also remain intact to produce normal RNFL birefringence. Thus, abnormal RNFL birefringence (in the presence of normal RNFL thickness) may signal states of RGC stress that include not only potentially abnormal production, modification, transport, and maintenance of tertiary structure but perhaps also a failure of proper three-dimensional spacing and alignment.
In summary, the results of this study demonstrate that SLP-derived measures of RNFL birefringence can change independently of RNFL thickness, as derived by OCT. Based on current hypotheses about the major source(s) of birefringence in the RNFL, this suggests that longitudinal comparisons between birefringence and RNFL thickness (e.g., by SLP and OCT or by polarization-sensitive OCT30 ) may be capable of signaling an intermittent state of axonal degeneration whereby internal structural elements such as MT or NF have become abnormal before changes in axonal caliber. It remains to be determined whether the results from this acute experimental manipulation will be applicable to the slower course of most disease states, though a similar differential time course has been observed after optic nerve crush.23 24 However, in diseases with a substantially slower course such as glaucoma, gliotic changes within the RNFL32 are likely to complicate measurement of this relationship. It is nonetheless interesting that Mohammadi et al.33 have recently shown SLP-derived measures of RNFL birefringence were independent predictors of future vision loss in glaucoma suspects who began the study with normal SAP visual fields, regardless of their age, IOP, or optic disc appearance.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 11, 2007; revised August 20, 2007; accepted November 14, 2007.
Disclosure: B. Fortune, None; L. Wang, None; G. Cull, None; G.A. Cioffi, Carl Zeiss Meditech Inc. (F)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Brad Fortune, Devers Eye Institute, Legacy Health System, 1225 NE Second Avenue, Portland, OR 97232; bfortune{at}deverseye.org.
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