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(Investigative Ophthalmology and Visual Science. 2004;45:144-152.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0687
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Model of Endothelin-1–Induced Chronic Optic Neuropathy in Rat

Balwantray C. Chauhan,1,2,3 Terry L. LeVatte,1,3 Christine A. Jollimore,1,4 Paula K. Yu,5 Herbert A. Reitsamer,6,7 Melanie E. M. Kelly,1,2,4 Dao-Yi Yu,5 François Tremblay,1,2,3 and Michele L. Archibald1,3

1From the Retina and Optic Nerve Research Laboratory, and the 2Departments of Ophthalmology, 3Physiology and Biophysics, and 4Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada; the 5Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Australia; and the 6Departments of Physiology and 7Clinical Pharmacology, University of Vienna Medical School, Vienna, Austria.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To describe a model of chronic endothelin (ET)-1 administration to the optic nerve and evaluate its effect on retinal ganglion cell (RGC) and axon survival in rat.

METHODS. Osmotic minipumps were surgically implanted in one eye of 113 Brown Norway rats to deliver 0.05, 0.10, 0.20, or 0.40 µg ET-1 per day (3.3, 6.7, 13.4, and 26.8 µM, respectively), or balanced salt solution (BSS) to the immediate retrobulbar optic nerve; the fellow untreated eye served as the control. Before pump implantation, RGCs were retrogradely labeled with fluorochrome. Animals were killed at 21, 42, or 84 days. RGC survival was expressed as the ratio of RGC counts in experimental versus control eyes in wholemounted retinas, whereas axon survival was expressed similarly from electron micrographs of the optic nerves. Serial optic disc changes were evaluated using scanning laser tomography. The effect of ET-1 (3 µL topical application of 10-5 M) on blood flow in the surgically exposed optic nerve was measured using laser Doppler flowmetry in a separate group of five animals.

RESULTS. ET-1 led to a mean reduction in optic nerve blood flow of 68%. There were no significant differences in RGC survival among the four ET-1 doses used in this study. Pooled across all ET-1 doses, RGC survival decreased incrementally at 21, 42, and 84 days (P < 0.001; mean ± SD, 0.77 ± 0.25, 0.60 ± 0.27, and 0.50 ± 0.26, respectively) and was statistically significantly lower at each time point than in the BSS-treated animals. The axon survival data also showed a similar time-dependent loss. Only one of 21 animals showed significantly increased disc cupping, and there was no relationship between RGC survival and change in cupping.

CONCLUSIONS. Chronic administration of ET-1 to the rat optic nerve results in a time-dependent loss of RGCs and their axons without apparent change in optic disc topography.

Open-angle glaucoma is a disease characterized by loss of retinal ganglion cells (RGCs), cupping of the optic disc, and defects in the visual field. Intraocular pressure (IOP) is the major known risk factor that produces glaucoma1 and glaucoma-like damage to the optic nerve and RGCs in experimental primate models2 3 4 5 ; however, other factors may interact with IOP to modulate its effect on the optic nerve. There is evidence to suggest that disturbances of blood flow in the optic nerve head may be one such factor,6 7 8 9 though direct evidence to support a putative role for ischemia in clinical glaucoma is not abundant.

Several research teams have used experimental in vivo models of ischemia to induce RGC death. The most widely used models are acute and involve either elevation of IOP to levels exceeding central retinal artery pressure10 11 or ligation of the central retinal artery12 or ophthalmic artery.13 In recent years, a model of endothelin (ET)-1–induced chronic optic nerve ischemia has been described by Cioffi et al.14 and Orgül et al.,15 first in the rabbit and subsequently in the rhesus monkey.16 ET-1 is a potent vasoactive peptide17 that reduces retinal,18 choroidal,19 and optic nerve head blood flow20 ; however, the effects of intravenous ET-1 on optic nerve head blood flow have shown conflicting results.20 21 The vasoconstrictive effects of ET-1 have been described in isolated porcine ciliary arteries22 and porcine and human retinal arterioles,23 where, at a given dose, extraluminal administration produces a more potent vasoconstriction than intraluminal administration.23 The role of ET-1 in human glaucoma is not clear; however, a recent study has shown that whereas baseline plasma ET-1 levels were similar between patients with glaucoma and healthy control subjects, only patients showed elevated plasma ET-1 after cold-induced vasospasm.24

In the model described by Cioffi et al.14 a small volume of ET-1 is delivered to the retrobulbar optic nerve through a sub-Tenon’s catheter connected to an osmotic minipump located subcutaneously above the eye. Orgül et al. reported a 36% and 38% reduction of blood flow, respectively, in the treated monkey16 and rabbit15 optic nerve head. An increase in optic nerve head cupping, as measured with scanning laser tomography, was also described.15

Although nonhuman primate models of glaucoma and other optic neuropathies best approximate the corresponding clinical entities, many researchers are using the relatively inexpensive rat models.25 26 27 28 29 The objective of this study was to modify the model described by Cioffi et al.14 for the rat, by using a surgical approach. We present the effect of ET-1 on optic nerve blood flow, RGC and axon survival, and optic disc topography.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Brown Norway rats (250–300 g) were housed in a 12-hour light–dark cycle environment and given food and water ad libitum. All procedures complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and ethics board approval was obtained from the Dalhousie University Committee on Laboratory Animals. Animals were anesthetized for surgery with a ketamine-xylazine-acepromazine cocktail and killed with an overdose of pentobarbital sodium (340 mg/mL). Both drugs were administered intraperitoneally.

Surgical Procedures
One week after acclimation, a sponge (Gelfoam; Pharmacia & Upjohn, Uppsala, Sweden) soaked with the neurotracer fluorochrome (2% Flurogold; Fluorochrome Inc., Denver, CO) was placed over the superior colliculi after the overlying cortex was aspirated, to label the RGC cell bodies by retrograde transport. Approximately 1 week later, an osmotic minipump (Durect Corp., Cupertino, CA) was implanted in the skinfold of the neck through a small incision. The optic nerve was carefully exposed by blunt dissection through an incision over the orbital ridge. A small hole was then drilled in the orbital ridge. One end of a silastic delivery tube was connected to the minipump, and the other end was channeled through the hole in the orbital ridge, where it was glued securely in place. The end of the delivery tube was located approximately 1 mm behind the globe and just above (but ensuring no contact with) the optic nerve. Intraoperative analgesia (0.02 mg/kg buprenorphine hydrochloride; Buprenex, Animal Resource Centre, McGill University, Montreal, Quebec, Canada) was administered intraperitoneally approximately midway through the surgery. The incision was sutured after application of a topical antibiotic (100 µg/mL gentamicin; Sigma-Aldrich Canada, Oakville, Ontario, Canada).

We used minipumps that delivered the perfusate at the rate of 0.25 µL/h for 28 days. We used 0.05, 0.1, 0.2, or 0.4 µg/d of ET-1 (Peptides International, Louisville, KY) dissolved in 0.1 mM balanced salt solution (BSS; Invitrogen-Gibco, Gaithersburg, MD) corresponding to doses of 3.3, 6.7, 13.4, and 26.8 µM respectively, or 0.1 mM BSS only. In experiments exceeding 28 days, minipumps were replaced as necessary. The ET-1 doses were selected to cover and extend, at either end, the doses used previously in other species.15 30 The experimental procedure was performed on one eye only, whereas the fellow eye served as the untreated control.

Measurement of Optic Nerve Head Blood Flow
Before conducting the chronic experiments, the effect of ET-1 on optic nerve head blood flow was measured in a different set of animals, by a fiber optic–based laser Doppler flowmeter (ALF-21; Transonic Systems Inc., Ithaca, NY). Laser Doppler flowmetry provides three indices of perfusion derived from the frequency spectra collected from tissue illuminated with laser light: the number of moving blood cells, their mean velocity, and flux. The flux signal is the product of mean velocity and the number of moving blood cells and has been shown to correlate linearly with independent measures of blood flow in a variety of tissues.31 A detailed description of laser Doppler flowmetry and its validation have been provided elsewhere.31

The optic nerve was first exposed as described earlier. The probe (Type NS, diameter 0.58 mm, length 40 mm, fiber separation 0.15 mm; Transonic Systems Inc.) was attached to a stereotaxic micromanipulator (SAS-4100; Bioanalytical Systems Inc., West Lafayette, IN) and lowered through the incision site until it touched the optic nerve just behind the globe. The probe was angled approximately 45° toward the globe to ensure that the whole vascular bed of the optic nerve head was within the sampling volume of the probe. In two animals, after a stable baseline blood flow trace was obtained over several minutes, 3 µL of 10-11 M ET-1 was delivered to the optic nerve in the vicinity of the probe with a gas-tight syringe (Hamilton Company, Reno, NV). After blood flow was recorded for approximately 15 minutes, the entire area surrounding the probe tip was irrigated with BSS. A stable baseline was again obtained before delivering 3 µL of 10-9 M ET-1. The procedure was then repeated for ET-1 doses of 10-7, 10-6, and 10-5 M. In three additional animals, only the 10-5 M dose was evaluated. The sampling rate of the flowmeter was 100 Hz. The data were analyzed by software that accompanied the flowmeter (Flowtrace-P; Transonic Systems Inc.). The baseline and final flow measurements for each dose were sampled over a 1-second window (100 measurements) after a stable recording was noted.

Scanning Laser Tomography
Scanning laser tomography of the rat optic disc was performed with a modified scanning laser tomograph (Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering GmbH, Dossenheim, Germany) as described previously.32 Briefly, the modifications involved changes in the laser output and scanning angles, as well as the use of a microscope objective and a glass plano-concave contact lens. The device was mounted on an operating microscope stand (model OPMi 6; Carl Zeiss Meditec, Thornwood, NY) and maneuvered with a custom-built remote arm that allowed 4° of movement (horizontal, vertical, height, and rotation).

The rat was placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) and the pupils fully dilated with 1% cyclopentolate (Diopentolate; Dioptic Laboratories, Markham, Ontario, Canada). After the optic disc was centered in the image frame, a minimum of three images was obtained. After processing, a mean reflectivity and topography image was obtained for each session. Baseline images were taken immediately after implantation of the pump and follow-up images immediately before death.

Changes in optic disc topography were assessed with a previously described method.32 A circular contour line was drawn well outside the optic disc margin in the baseline image. This contour line was imported to subsequent mean images and checked for proper placement. In images in which the alignment was not accurate, the same size contour line was placed manually. The ratio of cup volume in the final image to that in the baseline image was computed as the index of change in cupping. The 5th and 95th percentiles of the distribution of change in cup volume, due only to variability, was obtained in a group of control animals from a previous study32 in which the image-acquisition protocol was identical.

Tissue Preparation
Animals were killed at 21, 42, or 84 days after minipump implantation. After enucleation and optic nerve sectioning (described later), the globe was fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline for 3 hours. The retina was carefully dissected, flattened with four radial cuts, placed on a glass slide, and coverslipped with antifade medium (Citifluoro; Mirivac, Halifax, Nova Scotia, Canada). Fluorochrome-labeled RGCs were visualized using the UV-2A filter of the microscope (E800; Nikon, Mississauga, Ontario, Canada). Digital photomicrographs (640 x 480 µm) were taken centered at 1, 2, and 3 mm from the optic disc center in each quadrant after carefully focusing on the RGC layer.

After enucleation, the optic nerve was cut approximately 1 mm behind the globe and the nerve stump fixed immediately in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) overnight. The stump was rinsed with the buffer and placed in 1% OsO4 for 2 hours and then in 0.25% uranyl acetate for an additional 2 hours. The nerves were dehydrated with acetone and embedded in Epon Araldite (Mirivac). They were thin sectioned (100–130 nm), poststained with 2% uranyl acetate, and viewed on an electron microscope (EM300; Philips, Eindhoven, The Netherlands). After the center of the optic disc was located, three equidistant micrographs (83 x 58 µm) were taken in each quadrant of a prefixed rectangular grid. Longitudinal sections of retina were processed in the same way, sectioned (1 µm), stained with toluidine blue, and examined under light microscopy.

Quantification of RGC and Axon Survival
The fluorochrome-positive cells were counted manually from the micrographs. Because the fragments of dead RGCs are phagocytosed by microglia which then become fluorochrome-positive, we applied size (>10 µm) and shape (circular or oval, with an aspect ratio of 0.8–1.2) criteria, to ensure that only apparently surviving RGCs were counted. For each eye, RGC counts were averaged across each quadrant for the three retinal eccentricities, and RGC survival expressed as a ratio of the counts in the experimental to fellow control eyes.

Axon counts were performed manually from micrographs printed on 12.5 x 12.5-cm paper (12 per nerve) with a print magnification of x4800. Axon survival in the experimental eye was expressed as a ratio of the mean number of axons in the experimental to those in control eyes. Criteria for surviving axons included an intact myelin sheath, visible neurofilament, and absence of obvious swelling or shrinkage.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
A total of 118 animals were used in the study. Five animals were used for the blood flow experiments, 106 for the RGC survival analysis, and 24 for the axon survival analysis; 21 had scanning laser tomography performed. The retinas of seven animals in the latter group were not fluorochrome labeled; hence, RGC counts were not available.

In the preliminary optic nerve head blood flow experiments, all five animals received the 10-5 M ET-1 dose, whereas two animals received the range of doses. The mean baseline flow before 10-5 M ET-1 was 25.58 (range, 19.02–37.72) arbitrary units [AU]. The final flow measurement was 7.59 (range, 5.50–11.53) AU, corresponding to a mean change of -67.64% (range, -50.29% to -85.41%; n = 5). The corresponding mean changes after the 10-11, 10-9, 10-7, and 10-6 M ET-1 doses were -0.54%, 2.26%, 0.84%, and -51.25%. The blood flow traces from one animal across a range of doses of ET-1 is shown in Figure 1 .



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  		FIGURE 1. Effect of various concentrations of 3 µL topically administered ET-1 on optic nerve head blood flow measured with laser Doppler flowmetry. Arrows: time point of drug delivery.

 
The retinas of animals treated with ET-1 showed loss of RGCs, as identified by the size and shape criteria used in this study (Fig. 2) . Bright punctate fluorochrome-positive staining, which is likely to indicate pyknotic bodies as the RGCs undergo cell death, were frequently seen. Also present were fluorochrome-positive cells that were not determined to be RGCs by the study criteria but probably activated microglia that had phagocytosed the RGC debris and taken up the fluorochrome. The optic nerve cross sections of the ET-1 animals showed correspondingly reduced axon counts (Fig. 3) . Although a detailed analysis of the thickness of retinal layers was not possible because the large majority of retinas were wholemounted, inspection of the available sections under light microscopy showed that retinas of the ET-1–treated eyes had fewer cells in the RGC layer; however, the other cell layers appeared to be unaffected (Fig. 4) .



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  		FIGURE 2. Fluorochrome-labeled retinal wholemounts of eyes treated with 0.2 µg/d ET-1 (top) for 21 (A), 42 (B), and 84 (C) days and fellow control eyes (bottom). The overall proportion of surviving RGCs in the treated eyes was 0.90 (A), 0.64 (B), and 0.41 (C). In addition to a reduced number of RGCs, the treated eyes showed fluorochrome-positive cells that were not determined to be RGCs by the study criteria (arrows) and bright punctate bodies (arrowheads). Scale bar, 100 µm.

 


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  		FIGURE 3. Corresponding electron micrographs of the optic nerves of eyes shown in Figure 2 treated with ET-1 (top) and fellow control eyes (bottom). The overall proportion of surviving axons was 0.88 (A), 0.66 (B), and 0.38 (C). Scale bar, 10 µm.

 


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  		FIGURE 4. Light micrographs of the retina of an eye treated with 0.2 µg/d ET-1 for 42 days (left) and a normal control eye (right), stained with toluidine blue. There were fewer cells in the retinal ganglion cell layer (GCL); however, there were no other apparent differences in the other retinal layers. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRS, photoreceptor inner and outer segments. Scale bar, 25 µm.

 
Of the 106 animals in whom RGCs were fluorochrome labeled, 12 (11%), 20 (19%), 35 (33%), and 20 (19%) were treated with 0.05, 0.1, 0.2, and 0.4 µg/d of ET-1, respectively, whereas 19 (18%) were treated with BSS. The proportion of surviving RGCs at 21, 42, and 84 days after minipump implantation containing 0.05, 0.1, 0.2, and 0.4 µg ET-1 per day is shown in Figure 5 . The mean ± SD RGC survival in the BSS-treated animals at 21, 42, and 84 days was 0.89 ± 0.27 (n = 14), 1.04 ± 0.32 (n = 4), and 0.97 (n = 1), respectively. The data from the BSS-treated animals were pooled and are also shown in Figure 5 . The differences in RGC survival among the four ET-1 and BSS treatment groups did not reach statistical significance at the 21-day point (P = 0.226, ANOVA); however, they were significantly different at the 42-day (P = 0.011, with post hoc differences between the BSS-treated and the 0.2 µg ET-1/d treated animals, Tukey-B test) and 84-day (P < 0.001, with post hoc differences between the BSS-treated and the 0.05, 0.2, and 0.4 µg ET-1/d treated animals) time points. There were no significant differences in RGC survival among the four ET-1–treated animal groups at any of the time points, even after the BSS-treated animals were removed from the analysis (P > 0.372).



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  		FIGURE 5. Mean proportion of surviving RGCs at 21, 42, and 84 days as a function of ET-1 dose. Error bars, ± SEM. RGC survival in BSS-treated animals is shown as mean (solid line) ± SEM (shaded area).

 
The RGC survival data pooled across all the ET-1–treated animals are shown as a function of time (Fig. 6) . The mean ± SD RGC survival at 21, 42, and 84 days was 0.77 ± 0.25 (n = 43), 0.60 ± 0.27 (n = 18), and 0.50 ± 0.26 (n = 26), respectively, and was significantly different from that in the BSS-treated animals (P = 0.030, P < 0.001 and P < 0.001, respectively). These incremental differences in RGC survival in the ET-1–treated animals with time were also statistically significant (P < 0.001).



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  		FIGURE 6. Box plots showing proportion of surviving RGCs (left) as a function of time pooled across all doses of ET-1. Tails: 90th and 10th percentiles. Boxes: 75th, 50th, and 25th percentiles. ({blacksquare}) Means.

 
Electron micrographs of the optic nerve were available in 24 animals (8 each at the 21-, 42- and 84-day time points). Because of the relatively small number of animals and the apparent lack of effect of dose on RGC survival, results in the animals across each time point were pooled. The mean ± SD axon survival at 21, 42, and 84 days was 0.82 ± 0.23, 0.71 ± 0.34, and 0.61 ± 0.35, respectively. These data show a mean time course of axon survival similar to that observed for RGC survival.

There appeared to be no preferential RGC loss with retinal eccentricity. Although there was a decrease in RGC survival with increasing time, at any given time point it was not significantly different at 1, 2, or 3 mm from the optic disc center (P > 0.537; Fig. 7 ).



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  		FIGURE 7. Box plots showing proportion of surviving RGCs at 21, 42, and 84 days as a function of retinal eccentricity pooled across all doses of ET-1. Tails: 90th and 10th percentiles. Boxes: 75th, 50th, and 25th percentiles. ({blacksquare}) Means.

 
The distribution of the change in cup volume is shown in Figure 8 . Only one animal had an increase in cup volume that was outside the 95th percentile of retest variability, and one animal had a borderline decrease in cup volume. In the 14 animals in which RGC survival counts were also available, there was no statistically significant relationship between change in cup volume and RGC survival (Spearman’s r = 0.407, P = 0.149). None of these animals showed a statistically significant increase in cup volume, often in spite of substantial loss of RGCs (Fig. 9) .



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  		FIGURE 8. Left: distribution of ratio of final to baseline cup volume in animals that had serial scanning laser tomography examinations. Right: relationship between ratio of final to baseline cup volume and proportion of surviving RGCs. Shaded area represents the central 90% confidence interval of the cup volume ratio in a control group of animals with no intervention.

 


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  		FIGURE 9. Color-coded baseline (top left) and final (bottom left) optic disc images of an eye treated with 0.1 µg/d ET-1 showing no apparent change in optic disc cupping (ratio of final to baseline cup volume, 1.12). Fluorochrome-labeled retinal wholemounts of the corresponding eye (top right) and fellow control eye (bottom right). The overall proportion of surviving RGCs in the treated eye was 0.60. Scale bar, 100 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Experimental models of chronic optic nerve damage often result in considerable variation in the index of damage, such as RGC or axon survival. Therefore, investigating whether a given intervention to either ameliorate or advance the damage is effective, studies typically require an appropriately large sample of animals. Many laboratories have chosen to focus on cost-effective rodent models in which appropriate sample sizes are possible to allow such experiments to take place, at least as a first step before experiments in the more relevant primate models.

The objective of this study was to describe a surgical modification in rat, of a model of ET-1–induced chronic optic nerve ischemia described by Cioffi et al.14 and report on its effect on neuronal survival. Our results showed that chronic doses of ET-1 from 0.05 to 0.4 µg/d (3.3–26.8 µM) led to RGC losses of 23% at 21 days, 40% at 42 days, and 50% at 84 days. Although the number of animals that had available RGC axon counts was smaller, the corresponding values of 18%, 29%, and 39% also show a similar rate of loss.

Although we demonstrated statistically significant, time-dependent loss of RGCs, we were not able to find differences in RGC loss across the doses used in this study. This finding may be explained by the fact that on the logarithmic (molar) scale, the range of doses used was quite narrow, and had we used a broader range, dose-dependent differences may have emerged. An alternative possibility is that our study was statistically underpowered to detect dose-dependent differences in RGC survival. Based on a two-sample group difference and the typical standard deviation in the RGC survival rates, approximately 100 animals per group would have been necessary to detect a 10% difference in RGC survival rate with 80% power. The corresponding number for a 20% and 30% group difference are 25 and 11 animals per group. Although there appeared to be no trend of increasing damage with increasing dose at either of the time points (Fig. 5) , we cannot rule out the possibility that less than 20% differences among the treatment groups that were not detectable by this study could have existed.

In our acute experiments ET-1 concentrations of up to 10-7 M did not have an effect on optic nerve head blood flow; however, higher concentrations (10-6 and 10-5 M) led to a pronounced reduction. The ET-1 concentrations that produced RGC loss in the chronic experiments were of the same order (3.3–26.8 x 10-6 M) as those that reduced optic nerve head blood flow. We acknowledge, however, that the link between findings in acute studies and chronic ones, in which the effects of long-term ET-1 delivery on optic nerve circulation, ET-B receptor–mediated nitric oxide release33 with potential vasodilatory effects, long-term ET-1 diffusion to the nerve, and possible ET-1 tachyphylaxis are not known, should be made with caution.

We attempted to measure the long-term changes in optic nerve head blood flow with both noninvasive and invasive techniques to correlate these findings with RGC loss but were not successful. Scanning laser Doppler flowmetry has been used to measure optic nerve head blood flow in humans34 and monkeys35 ; however, our attempts in rat were unsuccessful. That retinal vessels almost completely cover the disc surface means that measurements in the underlying tissue cannot be made. Although microspheres have been used successfully in monkey36 and rabbit37 optic nerve head, the number of microspheres harvested from the rat optic nerve were insufficient for meaningful interpretation. We experimented with radiolabeled desmethylimipramine, which has been used to measure blood flow in rat sciatic nerve38 ; however, we found the variability of the measurements to be unacceptable. Furthermore, we were often unable to find differences between measurements in an optic nerve in which the ipsilateral ophthalmic artery was ligated and the contralateral control side. Finally, remeasurements of optic nerve blood flow using the laser Doppler technique described in this study before killing the animal to compare with baseline are probably not meaningful. In addition to relocating the probe in exactly the same position, any changes in the scattering properties of the tissue, which is inevitable given the invasiveness of the procedures, would make the measurements difficult to interpret. Although we have shown that ET-1 clearly reduced optic nerve head blood flow in acute studies, we recognize the limitation that a correlation between longitudinal changes in blood flow and RGC loss could not be made.

We were not able to document any optic disc changes with scanning laser tomography with this model of optic neuropathy. The absence of disc cupping in spite of substantial RGC loss was also noted by us in a model of IOP-induced optic neuropathy; however, cupping was nearly always present when less than 50% of axons survived.32 That only one of the 21 animals showed a change in cup volume that was outside the normal range (which could be an expected false-positive finding, given the sample size and the variability limits) and that there was no relationship between change in cup volume and RGC survival suggests strongly that ET-1–induced optic neuropathy does not cause topographical optic disc changes. These findings contrast with those of Orgül et al.,15 who reported a decreased neuroretinal rim in rabbits treated similarly with ET-1; however, there are substantial structural differences between the rabbit and rat optic nerve head that may explain these differences.

Optic disc surface changes and pallor are sometimes reported in the end-stage arteritic anterior ischemic neuropathy39 40 41 ; however, the nature of the changes are different from those seen in glaucoma.41 42 In contrast, nonarteritic anterior ischemic neuropathy does not result in cupping.41 43 Finally, there is little evidence of extracellular matrix changes in the optic nerve head when axons are lost by optic nerve transection,44 45 which is in sharp contrast to that found after axonal loss in clinical46 47 or experimental models of primate44 48 49 50 or rat glaucoma.51 These previous studies and our earlier work32 where there was evidence of both in vivo topographical disc changes and morphologic changes in IOP-induced damage with a degree of RGC or axonal loss similar to that in this study suggest that optic disc cupping may not be a consequence of axonal loss, but rather that it may be modulated by the mechanical action of IOP, irrespective of the origin of RGC loss.

The mechanism whereby exogenous ET-1 results in RGC loss is not known.52 Recent studies have shown that cultured human optic nerve astrocytes proliferate after exposure to ET-1 through ET-A and -B receptor activation.53 It is possible that the normal glia-neuron interaction may be disrupted by the reactive astrocytosis and exacerbate the rate of neuronal loss. Other evidence suggests that intravitreal administration of ET-1 interferes with anterograde axonal transport.54 It is likely that in addition to ischemia, there is involvement of multiple factors that precipitate in neuronal loss and whose roles remain to be investigated by using a variety of complementary in vivo and in vitro techniques.

In summary, we have described a model of chronic ET-1–induced optic neuropathy in the rat in which there is a time-dependent loss of RGC and their axons. Unlike IOP-induced optic neuropathy,32 in the rat, this type of insult does not lead to optic disc cupping. Further work is now under way to determine whether eyes with chronic ischemia-induced neuropathy can develop optic disc cupping at lower levels of experimentally elevated IOP.


   Footnotes
 
Supported by Grant MOP-57851 from the Canadian Institutes of Health Research (BCC) and funds from the Austrian Science Foundation FWF J1866-MED (HAR) and the National Health and Medical Research Council of Australia (D-YY).

Submitted for publication July 4, 2003; revised August 26, 2003; accepted September 6, 2003.

Disclosure: B.C. Chauhan, None; T.L. LeVatte, None; C.A. Jollimore, None; P.K. Yu, None; H.A. Reitsamer, None; M.E.M. Kelly, None; D.-Y. Yu, None; F. Tremblay, None; M.L. Archibald, None

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: Balwantray C. Chauhan, Department of Ophthalmology, Dalhousie University, 2nd Floor Centennial Building, Queen Elizabeth II Health Sciences Centre, Halifax, NS, Canada B3H 2Y9; bal{at}dal.ca.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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