Animal models on the effects of chronic elevation of IOP on the optic nerve head are providing important information on the structural changes in both the optic nerve and retina. This information will ultimately lead to a better understanding of the pathogenesis and treatment of the glaucomas. The objective of this study was to characterize the relationship between the level of IOP and the ensuing structural and functional damage.
Our data show that optic disc cupping, as measured with noninvasive scanning laser tomography, and changes in the ERG were dependent on the peak elevation of IOP in the experimental eye compared with that in the fellow untreated eye (peak ΔIOP). These changes were also dependent on the product of the IOP elevation (compared with the fellow eye) and days of IOP elevation (ΔIOP integral). Optic nerve axonal loss additionally depended on these IOP parameters. We were not able to show a relationship between the days of IOP elevation and either of the structural or functional parameters, suggesting that there may an IOP threshold below which measurable damage does not occur.
The IOP in normal eyes of conscious Brown Norway rats is approximately 20 mm Hg. Our results show that if peak ΔIOP exceeds 15 mm Hg (∼35 mm Hg, or a factor of 1.75 above normal), extensive axonal loss (mean, 69.2%), optic disc cupping, and electrophysiological loss usually occur. If peak ΔIOP exceeds 20 mm Hg, that is a factor of 2 above normal, profound structural (with a mean of 76.7% axonal loss), and electrophysiological losses occur. It should be noted that the equivalent thresholds for mean IOP elevation would be substantially less. The relationship between peak ΔIOP and axonal loss is in agreement with Morrison et al.
29 In another study, the same group reported 100% nerve degeneration when mean ΔIOP was higher than 20 mm Hg.
25
Compared with the present study and that by Morrison et al.,
29 RGC losses for equivalent elevations in IOP are more modest in a rat model in which argon laser irradiation of the trabecular meshwork was used
31 and in a model in which argon laser photocoagulation of the episcleral and limbal veins was used.
27 Our results diverge, however, from those obtained in studies in which the thermal cautery model, as described by Sharma et al.,
26 was used. Their group reported approximately 35% RGC loss in animals in which IOP was increased by an average factor of 2 for 42 days.
37 At 42 days, using a similar model, Sawada and Neufeld
33 reported approximately 11% and 6% RGC loss in the peripheral and central retina, respectively, in experimental eyes with an IOP 1.7 times higher than in fellow control eyes. At 182 days (6 months), the respective RGC losses were 39% and 12%, whereas the IOP was 1.5 times higher than in fellow control eyes. Finally, using the cautery model with application of 5-fluorouracil, Mittag et al.
38 showed only qualitative patchy ganglion cell losses in eyes with IOPs elevated by a factor of 2 to 3 for 3 to 4 months. It is likely that methods of elevating IOP, strain of rats, methods of measuring IOP, state of consciousness of the animal during measurements, and methods of quantifying axonal or ganglion cell loss have contributed to the differences in results observed in the literature. Furthermore, unlike the present study, these studies did not report the relationship between IOP and damage on a individual basis, but rather averages of IOP and neural damage parameters across groups.
To the best of our knowledge this is the first study to show progressive cupping in the same animal in a rat model of IOP-induced optic neuropathy. Scanning laser tomography allows monitoring of structural changes and measurements at different time points. Our results show that progression of optic disc cupping can occur relatively quickly, suggesting that these changes may develop after an IOP threshold has been reached. That cupping was not noted with scanning laser tomography until there was considerable axonal loss indicates either that the technique is not sensitive enough or that in this model cupping is primarily a mechanical phenomenon related to the level of IOP. Caution should be exercised in extrapolation of this finding to other species, because in the rat the large area occupied by the blood vessels on the optic disc and their fanned distribution on the optic disc and peripapillary retina may provide considerable structural support to the disc surface. In other species, cupping may occur with more modest axonal loss and may therefore be detectable earlier by imaging techniques. The normal rat disc also has little or no physiological disc cupping and therefore development of even a small degree of cupping would result in large changes in the cup volume change ratio as observed in this study.
Inspection of the optic disc images and histologic sections (
Figs. 2 3 , respectively) shows evidence of expansion of the scleral canal. This phenomenon has been described previously in monkeys with experimentally IOP elevation
39 and may be due to increased scleral wall stress at the posterior pole.
40 Estimating the expansion of the scleral canal and its dependence on IOP can only be performed reliably with histologic analysis since outlining the scleral canal in topographic analysis is not accurate, because of the nature of the rat optic disc. For this reason we placed the contour line well outside the actual optic disc margin to avoid potential inaccuracies due to serial image misalignment or scleral canal expansion. Therefore, although what was defined as cup area and volume may not be accurate, this approach is better suited to detect changes in topography within the contour line and is less influenced by any size changes in the scleral canal.
The a-wave of the Ganzfeld ERG is classically thought to represent photoreceptor activity, whereas the b-wave reflects bipolar and Müller cell function.
41 These components of the ERG have been shown to be affected in some patients with glaucoma
42 43 which suggests either that, at least in some cases, glaucoma affects retinal cells besides RGCs or that there is a component of the ERG that reflects ganglion cell activity. Previous experimental work in rats has shown time-dependent changes in ERG parameters
38 44 ; however, the relationship between IOP and the ERG findings was not reported. We were not able to show systematic alterations in the a-wave with IOP elevation, which suggests that, at least functionally, the photoreceptor layer is unaffected in this model. Both the b-wave and ERG ratio, as defined in this study, were affected only at high IOP, suggesting loss of inner retinal function in these animals. Histologic evidence corroborates these electrophysiological findings
(Fig. 3) . The ERG ratio was generally unaffected with early or moderate axonal losses but was outside normal limits with extensive axonal loss when presumably damage had affected the inner retina. This was due to alterations in the b- and not the a-wave. However, because IOP is a covariate, it follows that the electrophysiological changes may occur as a result of IOP elevation itself and not solely as a result of neuronal loss.
In summary, our study shows that in an IOP-induced model of optic neuropathy, both cupping as measured by scanning laser tomography and ERG changes were strongly related to the peak IOP and IOP integral in the experimental eye, compared with the fellow untreated eye. These structural and function changes were independent of the days of IOP elevation, suggesting that they may be dependent on an IOP elevation threshold. Our data also show that these in vivo structural and functional parameters can be unaltered in spite of moderate levels of axonal loss.