Efficacy and Safety of Memantine Treatment for Reduction of Changes Associated with Experimental Glaucoma in Monkey, II: Structural Measures

Abstract

purpose. To determine, using anatomic measurements, whether daily oral dosing with memantine is both safe and effective to reduce the injury associated with experimental glaucoma in primates.

methods. Argon laser treatment of the anterior chamber angle was used to induce chronic ocular hypertension (COHT) in the right eyes of 18 macaque monkeys. Nine animals were daily orally dosed with 4 mg/kg memantine while the other nine animals received vehicle only. Measurements of intraocular pressure (IOP) from both eyes of all animals were made at regular intervals. Appearance of the optic nerve head, retinal vessels, and surrounding retina was documented with stereo fundus photographs obtained at multiple time points throughout the study. Measurements of optic nerve head topography were obtained from confocal laser scans made from animals with the highest IOPs at approximately 3, 5, and 10 months after elevation of IOP. At approximately 16 months after IOP elevation, animals were killed and histologic counts of cells in the retinal ganglion cell (RGC) layer were made.

results. Histologic measurements showed that, for animals with moderate elevation of IOP, memantine treatment was associated with an enhanced survival of RGCs in the inferior retina. Measurements of optic nerve head topography showed less IOP-induced change in memantine-treated animals. This effect was seen in measurements of both the cup and the neuroretinal rim. A comparison of these same histologic and morphologic measurements in normotensive eyes from the two treatment groups showed that memantine treatment was not associated with any significant effects on these eyes.

conclusions. Histologic measurements of RGC survival as well as tomographic measurements of nerve head topography show that systemic treatment with memantine, a compound which does not lower intraocular pressure, is both safe and effective to reduce changes associated with experimental glaucoma.

Electrophysiological evidence has been presented to show that oral treatment with memantine (1-amino-3,5-dimethyladamantane) was effective for the reduction of functional loss associated with experimental glaucoma in monkeys. ¹ This article reports on anatomic measurements made in these same animals in an effort to further ascertain the safety and efficacy of memantine treatment. Stereoscopic fundus photographs were made from both eyes of all animals at multiple times during the study. Using confocal scanning laser tomography, noninvasive measurements of optic nerve head topography were made at three time points. These measurements are correlated with visual field loss in clinical glaucoma studies ^{2 3 4 5 6 7 8 9 10} and also with the degree of optic nerve axonal loss in monkeys with experimental glaucoma. ¹¹ Since glaucomatous injury results in a loss of retinal ganglion cells (RGCs) in patients ^{12 13 14} as well as in monkeys with experimental glaucoma, ^{15 16 17 18} histologic measurements of retinal ganglion cell (RGC) survival were also made from retinal samples obtained from both eyes of all animals in each treatment group at the termination of the study. 

Methods

All procedures conformed to the guidelines in the Statement for Use of Animals in Ophthalmic and Vision Research provided by the Association for Research in Vision and Ophthalmology and were also reviewed by an institutional review committee. 

Animal Subjects

Eighteen young adult cynomolgous monkeys, Macaca fascicularis, were randomly divided into two groups and baseline measurements of IOP were made under light ketamine sedation (5 mg/kg, i.m.) using a pneumatonometer (Digilab, Norwell, MA). Daily oral dosing with 4 mg/kg memantine was begun in one group while animals in the other group received vehicle only. Argon laser cautery of the anterior chamber angle was performed on the right eyes of all animals in both treatment groups to induce chronic ocular hypertension according to the method originally described by Gaasterland and Kupfer. ¹⁹ IOP was measured under sedation with i.m. ketamine (5 mg/kg) using a Model RT pneumatonometer (Digilab, Norwell, MA) for IOP ≤ 45 mm Hg or a different pneumatonometer (Alcon, Fort Worth, TX) for IOP >45 mm Hg at regular intervals, indicated in Figure 1 , throughout the duration of the study (see Methods section in preceding paper ¹ for greater detail regarding IOP measurements). Blood samples were obtained from all animals at 2 months after the onset of oral dosing and at 2-month intervals thereafter. Immediately before the animals were killed, vitreous samples were also obtained from all eyes. The quantity of memantine in the plasma and vitreous samples was determined, in triplicate, using an internal standard method in combination with HPLC. Memantine-treated animals had blood and vitreal concentrations on the order of 1 μM for all measurement time points more than 2 months after laser treatment. For a detailed description of the procedure for measuring memantine levels in plasma and vitreous, as well as the results of those measurements, see the Methods section and Table 3 in the preceding paper. ¹  

Estimation of Mean IOP

For each animal, IOP was plotted as a function of time over the course of the study (approximately 16 months). Since HRT measurements were obtained at three time points, mean IOP was estimated for each time interval (t1 = 3 months, t2 = 5 months, and t3 = 10 months) by first integrating the area of this plot over the limits extending from the time at which the first elevation of IOP above baseline was obtained (t0; soon after laser treatment) to the time at which the HRT measurements were obtained (either t1, t2, or t3; see Fig. 1 in this article and Fig. 1 in the preceding article ¹ ). This integral was then divided by the number of days in that interval to yield an estimate of the mean IOP over the duration of each time period. For comparison with results of histologic measurements, mean IOP for each animal was estimated for the duration of the study by integrating the IOP plots from the peak IOP to the last IOP measure obtained immediately before sacrifice. 

Stereoscopic Fundus Photos

Simultaneous stereo pair fundus images were obtained using a stereo fundus camera (Nidek Inc., Fremont, CA) through pupils dilated to approximately 6 mm diameter with 1% tropicamide. Animals were anesthetized by i.m. injection of ketamine (10 mg/kg). Rigid contact lenses were used to provide an optimal optical surface for retinal imaging. Images were captured using Kodak Lumiere 100 ASA color slide film (Kodak, Rochester, NY) in combination with a flash level (intensity) of 3. 

Optic Disc Tomography

Optic disc morphology measurements were made using a confocal scanning laser opthalmoscope (Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering GmbH, Heidelberg, Germany). Image acquisition and processing have been described in detail previously. ^{11 20} Briefly, each image series was comprised of 32 transverse optic sections obtained at consecutive height planes over a scan depth of 1.5 to 2.5 mm. For each eye, three separate 15° images were taken and mean topography was determined using HRT software version 2.01 (Heidelberg Engineering GmbH). For each image, disc margins were manually outlined with the aid of stereo optic disc photographs obtained at approximately the same time. Image magnification errors were corrected using keratometric measurements of corneal anterior surface curvature. The standard reference plane was positioned at 50 μm posterior to the mean height of the optic disc margin contour over the temporal segment from 350° to 356°. Scans were made under general anesthesia induced with i.m. ketamine (10 mg/kg) in combination with paralysis maintained with continuous IV infusion of norcuronium bromide (0.04 mg/kg per h). Scans were obtained at approximately 3, 5, and 10 months after laser treatment to induce ocular hypertension as indicated in Figure 1 . Measurements were made on both eyes of ten animals including the five animals with the highest IOPs (OD) in each of the two treatment groups. Only eyes having the highest mean IOPs were measured because, due to the limited availability of the HRT instrument, only 10 animals could be followed and these eyes were expected to show the greatest change in nerve head morphology over the course of the study. Analysis of all scans was made by persons who were masked with regard to the treatment group to which each animal belonged. 

Histology

At the termination of the study, all animals were deeply anesthetized with a combination of ketamine (15 mg/kg), xylazine (1 mg/kg), and acepromazine (0.2 mg/kg) and transcardially perfused with heparinized saline, followed by a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) in phosphate buffer. The fixed retina/choroid was flat-mounted on a glass slide and 3-mm square samples were obtained from eight regions including one sample centered on the fovea (PF), three perimacular samples (1 to 3), and four samples from the periphery (4 to 7) as shown in Figure 2 . Note that for samples 1 to 7, the plane of section is orthogonal to the meridian which bisects that sample, whereas the plane of section for sample PF is orthogonal with the vertical meridian. All samples were paraffin-embedded for sectioning. For samples 1 to 7, ten radial sections of 7 μm thickness were obtained, beginning at the edge indicated by the heavy border, at 50 μm intervals and stained with hematoxylin/eosin. Cells in the ganglion cell layer (GCL) were manually counted over the entire 3 mm section for all ten sections. Glia and vascular cell nuclei were easily identified using size (<4 μ diameter) and morphologic (not round) criteria and were not counted. Perifoveal (PF) cell counts were made on ten sections from the region of highest cell density between 0.5 to 0.7 mm from the center of the foveal pit. Examples of PF sections obtained from the hypertensive (OD) and normotensive (OS) eyes of a vehicle-treated animal are shown in Figure 3 . Note that almost all the darkly stained nuclei in the RGC layer (GCL) are of similar size and appearance. Cell counts in the GCL from these perifoveal sections were made using a BIOQUANT imaging system and stereology software (R & M Biometrics, Inc., Nashville, TN). The software displayed an active image window and tagged objects that had been identified as cell nuclei according to the image analysis algorithm. This image was inspected by the operator and edited to include or exclude objects that did not meet the size and morphology criteria. For PF sections from eyes with severe RGC loss where the RGC layer was reduced to a single layer of cell nuclei, cells counts were made manually without the use of BIOQUANT. Using these methods, summed counts from all eight retinal sample regions totaled, on average, approximately 15,000 cells in the normotensive eyes of both treatment groups. All histologic analysis was performed by persons who were masked with regard to the treatment regimen for any animal. 

Statistical Analysis

The method of least squares ²¹ was used for linear regression analysis of data sets which express either histologic (Fig. 3) or morphologic measurements from the two treatment groups (vehicle or memantine) as a function of mean IOP. This method provided measurements of slope (m) and correlation coefficient (r) for each data set. The hypothesis that the slopes for regressions of the two treatment groups were equivalent was then tested using analysis of covariance with both treatment group and mean IOP as covariates. ²² In this model, either RGC counts or HRT measurements are the dependent variable while mean IOP and treatment group are independent variables. This comparison includes interaction between IOP and treatment group in the model. When data sets from the two treatment groups were compared in this manner, it was concluded that they were significantly different if the P value for the analysis of covariance was < 0.05. 

A Hochberg adjustment ²³ (“stepdown Bonferroni method”) was used to control for possible errors associated with comparisons of RGC counts from multiple regions of the retinas from the two treatment groups. Significance levels for P values obtained from the Student’s two-tailed t-test for comparisons of RGC counts from each sample region were adjusted according to this method. A model for Multivariate Analysis of Covariance ^{24 25} was used to control for possible errors associated with comparisons of multiple HRT measurements in the two treatment groups. An analysis of covariance with treatment, mean IOP, and interaction between mean IOP and treatment was performed jointly for either the five cup measurements or for the five neuroretinal rim measurements obtained from the two treatment groups. This analysis was made for measurements obtained at each of the three measurement time points. A significance level of P ≤ 0.05 was adopted for these comparisons. 

Results

Histology

Optic disc photographs and histologic sections from a vehicle-treated animal (M91; see Table 2 ) are shown in Figure 3 . The disc images were obtained at approximately 5 months after elevation of IOP (near HRT measurement time point t2) at which point the mean IOP history for the hypertensive eye was approximately 50 mm Hg. A single image from the stereo pair for the hypertensive (OD) and normotensive (OS) eye is presented in the top panels. Note the atrophic appearance of the disc and nasal deflection of the vessels in the hypertensive eye. Directly below each fundus photograph is a micrograph of a section from the PF sample region of that eye. Sections used for RGC counts in the PF region were obtained from that region where RGC density is highest (see Methods). This is readily apparent from inspection of the section from the normotensive (OS) eye, where the RGC layer is seen to consist of six or seven layers of RGC nuclei. A section from the identical retinal region in the hypertensive (OD) eye shows that ocular hypertensive injury has reduced the RGC layer to a single layer of nuclei. Note that while most of the perifoveal RGCs have been lost in this hypertensive eye, there is no gross evidence for loss of cells in either the outer nuclear layer (ONL; photoreceptor cells) or inner nuclear layer (INL; horizontal cells, bipolar cells, Mueller cells, and amacrine cells). This apparently selective loss of cells in the RGC layer was evident in histologic sections from all eight retinal sample regions. 

Counts for cells in the central (PF + 1 → 3) ganglion cell layer of the hypertensive eyes from both treatment groups were highly correlated with mean IOP as shown in Figure 4 . Cell counts obtained from PF or peripheral (samples 4 to 7) retina yielded results which were similar to those obtained for the central retinal counts (data not shown). A comparison of the linear regression plots for the two treatment groups shows that the relationship between mean IOP and ganglion cell loss is similar. In summary, this histologic measure of ganglion cell loss, summed over all eight retinal regions and obtained from all animals after approximately 16 months of ocular hypertensive injury, shows no evidence for a protective effect of memantine treatment. 

However, RGC counts were also compared from the four eyes of each treatment group whose mean IOP was moderately elevated to the range of 26 to 39 mm Hg. When compared to the eyes with the highest IOP, RGC loss would be expected to occur at a relatively slower rate in these eyes, thereby providing a better opportunity to see any protective effect of memantine treatment. A comparison of cell counts from either perifoveal (sample PF), perimacular (samples 1–3), peripheral (samples 4–7), superior (samples 1, 4, 5), and inferior (samples 2, 6, 7), retinal regions showed that cell loss in the memantine-treated group was less in the inferior retina (mean loss, 7%) than that seen in the vehicle-treated group (mean loss, 22%). Results from counts in the inferior retina of the two groups are illustrated in Figure 5 . There was no difference between the two treatment groups for counts obtained from the perifoveal, perimacular, peripheral, or superior retinal regions. A summary of the cell counts and mean IOP for the eight eyes represented in Figure 5 is provided in Table 1 , where P values (Student’s two-tailed t-test) for a comparison of measurements from the two groups are also listed. Table 1 also shows that the distribution of mean IOP values, obtained over the time course from T0 to the time of death, is similar among the two groups (detailed IOP histories for these eight eyes are included in Table 1 from the preceding paper ¹ ). Note that the difference for RGC counts in the inferior retina was significant at the P = 0.01 level. However, multiple retinal areas are compared and these measurements may not represent independent measurements of RGC loss. To avoid type one errors, the significance levels for the results in Table 1 were therefore adjusted according to the Hochberg method for multiple comparisons (modified Bonferroni method). According to this adjustment, the P value for comparison of the inferior retinal counts (sum of samples 2, 6, and 7) would need to be 0.008 or less to be regarded as statistically significant (nonadjusted confidence level of P = 0.05). Our obtained P value of 0.01 is only slightly greater than this value. RGC counts were also compared from each of the eight individual sample regions in the same moderate IOP animals of both treatment groups. Only sample region 6 showed a difference of the memantine-treated (OD/OS = 1.04 ± 0.02 SEM) and vehicle-treated (OD/OS = 0.82 ± 0.04 SEM) animals with a P value < 0.05 (P = 0.004, Student’s two-tailed t-test). This difference is also significant when compared to the P value obtained from Hochberg adjustment for multiple comparisons (P ≤ 0.006). Thus, a comparison of retinal cell counts from eyes with moderate IOP elevation showed that memantine treatment was associated with a significant preservation of cells in the RGC layer of the inferior retina. 

To determine whether memantine treatment was associated with any histologic signs of retinal toxicity, counts of cells in the ganglion cell layer of all normotensive (OS) eyes from the two treatment groups were studied. Figure 6 shows that RGC counts from the memantine-treated group were not significantly (P < 0.05) different from the vehicle-treated group. This was true for counts from all individual sample regions (PF, 1, … , 7) as well as for the sum of counts from all samples (PF + 1→7). Furthermore, inspection of the H/E stained sections from normotensive eyes of both groups showed no evidence for an effect of memantine treatment on the density or appearance of any other retinal layer/cell type (data not shown). 

Optic Nerve Head Morphology

Simultaneous stereo fundus photographs were obtained from both eyes of all animals at approximately 5, 10, 14, 20, 48, and 60 weeks after IOP elevation (T0 in Fig. 1 ). Stereo fundus photographs were used as an aid for manually outlining the disc margin in HRT scans (see Methods). These photographs were also examined for evidence of media opacities, disc hemorrhages, vascular nonperfusion, or any overt signs of ischemic insult to the optic nerve head and surrounding retina. Although pulsation of the central retinal vein was observed in several eyes at the first photograph session when IOP was highest, this was not seen at later time points and pulsation of the central retinal artery was never observed. Nor were disc hemorrhages ever observed in any eye at any time. The only vascular anomaly ever encountered was the characteristic nasal deflection of large vessels at the disks of eyes with advanced cupping. At no time was there any evidence of disc edema or media opacity in any eye. In both treatment groups, eyes having the highest IOP elevations (all animals included in Table 2 ) showed evidence of moderate to severe optic nerve atrophy (disc pallor) with considerable loss of the neuroretinal rim. Inspection of the superior and inferior peripapillary retina also showed evidence for loss of axons in the nerve fiber layer. These changes were less pronounced or not apparent in eyes having low or moderate IOP elevation (data not shown). Images obtained from a vehicle-treated animal (M91; see Table 2 ) with severe optic atrophy are shown in Figure 3 . 

Optic nerve head topographic measurements were derived from confocal laser tomographic scans made at approximately 3 (t1), 5 (t2), and 10 (t3) months after induction of chronic ocular hypertension. Scans were obtained from both eyes of the five animals having the highest mean IOP in each treatment group (Table 2) . Nerve head morphology was characterized by a series of measurements, summarized in Table 3 , which reflect properties of either the physiological cup or neuroretinal rim. Since nerve head morphology differed considerably from one animal to another but was typically similar among two eyes from the same animal, measurements for the hypertensive (OD) eye were normalized with respect to the measure obtained from the contralateral (normotensive) eye (OD/OS) and plotted as a function of mean IOP to characterize the effect of IOP on nerve head morphology. Figure 7 shows results obtained for measurements of the cup at approximately 5 months (t2) after elevation of IOP, while Figure 8 shows results of measurements of the neuroretinal rim obtained at the same time. In each of these plots, a steeper slope represents a greater effect of ocular hypertension on that measure. Note that for measurements of the cup (Fig. 7) , IOP elevation was associated with a relatively much greater effect in vehicle-treated animals than was seen in memantine-treated animals. However, measurements of the neuroretinal rim (Fig. 8) showed a more similar effect of IOP elevation in the two treatment groups. A summary of results from topographic measurements obtained at all three time points is included in Table 3 , which includes results from all animals from which tomographic scans were obtained. Note that for measurements obtained at all three time points, the slope for plots of cup measurements as a function of mean IOP for the vehicle-treated animals ranges from approximately 2 to 20 times greater than that obtained for memantine-treated animals. Results from an analysis of covariance (see Methods) showed that this difference was significant (P < 0.05) for five of the ten cup measurements at t2 and t3 but for none of the five cup measurements at t1. Of the fifteen neuroretinal rim measurements (five measurements at three time points), the slope for plots of the vehicle-treated animals was greater than the memantine-treated animals in ten of those measurements but this difference was statistically significant in only three cases. 

To control for the possibility of statistical error associated with making comparisons of multiple measurements of either the cup or neuroretinal rim, a multivariate analysis of covariance with treatment, mean IOP, and interaction of treatment and mean IOP was performed. Application of this model using all five cup measurements yielded P values of 0.9376, 0.0008, and 0.375 for measurements obtained at t1, t2, and t3, respectively. Application of the same model using all five neuroretinal rim measurements yielded P values of 0.9366, 0.3285, and 0.7689 for measurements obtained at t1, t2, and t3, respectively. Thus, differences for cup measurements obtained from the two treatment groups at t2 were highly significant. 

The results summarized in Figures 7 and 8 show clearly that ocular hypertension has profound effects on measurements of the optic nerve head. In memantine-treated animals, ocular hypertension is associated with much less effect on measurements of the cup than that seen in vehicle-treated animals, whereas effects of ocular hypertension on measurements of the neuroretinal rim are more similar in the two treatment groups. Although these results are summarized for the three measurement time points in Table 3 , it is difficult to tell from this summary how the individual measurements are changing over this time frame. For this reason, values (not normalized) for measurements obtained from the hypertensive eyes of the five animals in each treatment group were plotted at the three measurement time points in Figure 9 for measurements of the cup, and Figure 10 for measurements of the neuroretinal rim. The left-hand panels of Figure 9 show results for measurements of cup volume below the surface (A), cup shape (B), and cup area to disc area ratio (C), from vehicle-treated animals, while the right-hand panels show results for the same measurements from memantine-treated animals. A comparison of results from the two treatment groups shows clearly that memantine-treated animals had, on average, smaller measurements of cup volume and relative cup area. In fact, the average values for all measurements of cup volume or cup area in memantine-treated animals were smaller than those of the vehicle-treated animals at all three time points though these differences were not statistically significant (P > 0.05; Student’s two-tailed t-test). It is also clear that cup morphology, in almost all animals, was relatively stable over the time frame of these measurements; that is, most pressure-related change in cup morphology had already occurred by approximately 3 months after IOP elevation and there was relatively little change over the ensuing 7 months. A similar pattern is apparent in Figure 10 where it can be seen that, although some measurements vary somewhat more over the three time points, memantine-treated animals had, on average, larger measurements for rim area, rim volume, and RNFL thickness. RNFL thickness at t1 was the only measure of cup or rim morphology for which this difference was statistically significant (P < 0.05; Student’s two-tailed t-test). It is also clear that most IOP-related effects on rim morphology were apparent by 3 months after induction of chronic ocular hypertension. 

It is unlikely that an ocular hypertension-induced increase in OD disc size made any significant contribution to the disc measurements in this study. Disc size was very stable over the time course of our measurements from approximately 3 months to 10 months after induction of ocular hypertension. For any given hypertensive eye from either treatment group, disc size never varied by >7% over the three measurement time points. For any given normotensive eye from either treatment group, disc area never varied by >2% (data not shown). Any effect of ocular hypertension on disc size would be apparent from comparison of the hypertensive (OD) and normotensive (OS) disc area in the same animal. When OD/OS disc area measurements were averaged at each time point, the following values (average ± SD) were obtained for vehicle-treated animals: t1, 1.10 ± 0.13; t2, 1.11 ± 0.11; t3, 1.09 ± 0.12. Memantine-treated animals had average OD/OS disc area measurements equal to: t1, 1.02 ± 0.12; t2, 1.03 ± 0.12; t3, 1.03 ± 0.13. Although there was clearly a tendency for OD disc area to be greater than OS disc area in vehicle-treated animals, this difference was not significant (P > 0.05; Student’s two-tailed t-test) at any time point. There was also no significant difference between the OD/OS disc areas for the two treatment groups at any time. Thus, any contribution of an IOP-induced increase in OD disc size was likely small for the eyes in this study. 

Discussion

In the preceding companion article, ¹ electrophysiological measurements of RGC activity in the ERG responses of these same animals showed that eyes with the highest pressures suffered severe functional loss by approximately 3 months after IOP elevation and also showed that memantine treatment was associated with relatively less loss of function. This effect of memantine treatment, however, was observed only at the earlier measurement times and was not seen in recordings made immediately before sacrifice. It was proposed that, although memantine treatment had reduced the rate of RGC injury/loss, this effect could not be observed if the injury were allowed to progress over too long a study duration. Given the relatively rapid time course of functional loss in the eyes having the highest IOPs, it seems likely that even a relatively large effect of memantine treatment to decrease the rate of injury would not be evident after approximately 16 months of severe ocular hypertension. Results of RGC counts from these eyes (Fig. 4) are consistent with this model. A protective effect of memantine treatment was seen only when eyes with the highest IOP were excluded from the analysis (Fig. 5) . By excluding these eyes, the mean rate of injury may be reduced to levels that permit a protective effect to be observed even at 16 months. However, when the multifocal ERG measurements obtained immediately before sacrifice were analyzed similarly (exclusion of animals with the highest IOPs), any global or regional effect of memantine treatment could not be demonstrated (data not shown). This is likely due to the fact that the histologic evidence for protection was seen in perimacular and peripheral retina while the component of the ERG that is sensitive to RGC injury could be reliably measured only in the macula where RGC densities are greatest. ¹⁷ It is not clear why memantine treatment was associated with a relative preservation of RGC numbers in only the inferior retina. This observation may reflect a greater sensitivity of RGCs in this region to NMDA-type glutamatergic excitotoxic injury. However, a purely random variation in cell counts from either the superior or inferior sample regions cannot be completely excluded. 

The finding that memantine treatment was not associated with any histologic evidence for toxicity to RGCs (or any other retinal cell type) in the normotensive eyes (Fig. 6) is consistent with the observation, based on electrophysiological measurements of the ERG and VECP in these same animals (see preceding article ¹ ), that memantine treatment had no effect on normal function of the retina and central visual pathways. Thus, both functional and histologic/anatomic measurements show no evidence for a toxic effect of chronic systemic dosing with memantine. 

Memantine treatment was also associated with a relative preservation of normal optic nerve head morphology when compared to vehicle-treated animals; that is, elevated IOP was associated with changes in nerve head cup topography (i.e., cup area, depth, or volume) which were smaller in memantine-treated animals when compared to changes seen in vehicle-treated animals (Fig. 7) . When individual measurements of the cup were compared, this effect of memantine treatment was significant (P < 0.05) for half of the cup measurements at 5 and 10 months after induction of ocular hypertension (Table 3) . This memantine treatment effect was highly significant (P = 0.0008) at the 10-month measurement point if a model for multivariate analysis of all cup measurements jointly was applied to control for statistical errors associated with multiple comparisons. Although the slope of IOP-induced change for measurements of the neuroretinal rim from the memantine-treated group was smaller than that obtained from vehicle-treated animals at most time points (Table 3) , this difference was statistically significant in only three cases for comparison of individual measurements obtained at t2 or t3. When multivariate analysis was performed jointly on all measurements of the neuroretinal rim, there was no significant difference (P ≤ 0.05) between measurements from the two treatment groups at any time point. 

Nerve head measurements were obtained at approximately 3, 5, and 10 months after IOP elevation. Over this time frame, there was relatively little change in nerve head morphology in most eyes (Figures 9 and 10) . Most of the ocular hypertension related effects on measurements of either the cup or neuroretinal rim were apparent by approximately 3 months (t1) after IOP elevation and were relatively stable over the ensuing 7 months (t2, t3). At each of the three measurement times, memantine-treated ocular hypertensive eyes had, on average, smaller cup sizes and greater measurements of neuroretinal rim tissue than hypertensive eyes from vehicle-treated animals. Of course, this comparison does not consider the fact that the distribution of mean IOPs was somewhat different in the two treatment groups. However, this observation is consistent with results which do show a smaller effect of mean IOP on disc morphology in memantine-treated animals (see Table 3 ). 

It is interesting to consider why memantine treatment might have such an apparently large effect in preserving cup topography but relatively less effect on the neuroretinal rim. The results of histologic measurements from both treatment groups show that there were only small differences in the total number of surviving RGCs at 16 months after IOP elevation. ERG measurements of RGC function also suggest that memantine treatment afforded only modest protection that was limited to the first 5 months of IOP elevation. These functional measurements do not provide information about structural changes that may follow functional changes by many months. For example, an RGC may be nonfunctional to the extent that it no longer contributes to electrophysiological measurements of function (or to vision) and is in fact destined to die; and yet its axon and associated supportive tissue may persist for some period. At the last HRT measure (t3, 10 months after IOP elevation), the axons of otherwise dysfunctional, dying, or dead RGCs may still be present at the optic nerve head. This dysfunctional tissue may survive (persist) for longer periods of time in memantine-treated eyes and contribute to nerve head morphology in ways that result in relative preservation of cup morphology but less so to preservation of morphology of the neuroretinal rim. According to this scheme, the effect of memantine to preserve cup morphology might have been reduced or absent immediately before the end of the study had measurements been made at this later time (approximately 6 months after t3). Alternatively, it is possible that memantine treatment acted, in addition to any direct effect on RGCs, to preserve connective tissue associated with the lamina cribrosa, scleral canal, or vascular elements of the nerve head. These effects could directly affect the amount of tissue at the nerve head or indirectly affect cup morphology by increasing the resistance of the nerve head tissue to mechanical stresses associated with elevated IOP—effects which could favor preservation of cup morphology but have less effect to preserve morphology of the neuroretinal rim. 

The results presented here are consistent with a conclusion that memantine treatment is associated with a reduction of both histologic and morphologic measurements of ocular hypertensive injury but no evidence for an effect of memantine treatment on normal eyes. This conclusion is also consistent with results from functional measurements which were reported in the preceding paper. ¹  

 Submitted for publication June 5, 2003; revised December 21, 2003 and March 2, 2004; accepted March 17, 2004.

 Disclosure: W.A. Hare, Allergan, Inc. (E); E. WoldeMussie, Allergan, Inc. (E); R.N. Weinreb, (C, R); H. Ton, Allergan, Inc. (E); G. Ruiz, Allergan, Inc. (E); M. Wijono, Allergan, Inc. (E); B. Feldmann, Allergan, Inc. (E); L. Zangwill, None; L. Wheeler, Allergan, Inc. (E)

 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.

 

Figure 1.

 

Average IOP history for the laser-treated hypertensive (OD) eyes of all 18 animals in both treatment groups. Measurements from all animals were averaged and are plotted with the SD at each measurement point. Note that data points represent the times at which IOP measurements were obtained from both eyes of all animals in the two treatment groups and thus provide a record for the timing and frequency of IOP measurements during the study. The timing of the HRT measurements is indicated at approximately 3, 5, and 10 months post-IOP elevation (t1, t2, t3). Retinal samples for histologic analysis were obtained at approximately 16 months post-IOP elevation. Stereo optic nerve head photographs were obtained at multiple times over the course of the study. Individual IOP histories for all 18 animals of the study may be found in Table 1 of the preceding companion paper. ¹

Figure 2.

 

Location of retinal samples for histologic analysis. Each sample was 3 mm square and cut with the use of a transparent template from the fixed flat-mounted retina-RPE-choroid. The perifoveal (PF) sample was centered on the fovea. Samples 1 to 3 were located on the horizontal and vertical meridians from 3.5 to 6.5 mm from the fovea, while samples 4 to 7 were located on the oblique meridians from 8.5 to 11.5 mm from the fovea. Sections were cut from either the inferior edge (sample PF) or the edge facing the fovea (samples 1 to 7) as indicated by the heavy border. The dashed circle indicates the position of the optic nerve head (ONH).

Figure 3.

 

Fundus images from the hypertensive (OD) and normotensive (OS) eyes of a vehicle-treated animal (M91). Images were obtained at approximately 5 months after elevation of IOP. Mean IOP, measured from the time of initial IOP elevation (T0) to the time the image was obtained (t2) was equal to approximately 50 mm Hg (see Table 2 ). The atrophic appearance and nasal deflection of the large vessels of the nerve head is characteristic of eyes having the highest mean IOPs. Micrographs shown directly below the fundus images are hematoxylin/eosin-labeled sections from the perifoveal retinal sample region obtained from the same eye shown in the fundus image above. Calibration bar: 100 μ.

Figure 4.

 

RGC survival is highly correlated with mean IOP. Normalized (OD/OS) RGC counts from central retina (samples PF + 1 → 3) were summed and are plotted as a function of mean OD IOP for animals in both treatment groups. The slopes and correlation coefficients for the linear regression plots are −0.035 and 0.83, respectively, for the vehicle-treated group (filled squares, heavy line) and −0.022 and 0.86, respectively, for the memantine-treated group (open circles, light line). Analysis of covariance yielded P = 0.28. There are only seven data points for the vehicle-treated group since one animal was lost before sacrifice and one retina from the normotensive (OS) eye of another animal was lost during histologic preparation.

Figure 5.

 

Summary of normalized (OD/OS) RGC counts from the inferior retina (sum of counts in samples 2, 6, and 7; see Fig. 2 ) for the four eyes with moderate IOP elevation in each of the two treatment groups. RGC counts from all retinal regions are summarized for both treatment groups in Table 1 . The sum of RGC counts from these three retinal sample regions was, on average, approximately 2600 for the normotensive (OS) eyes of both treatment groups. See Methods for details regarding counts of cells in the RGC layer and Figure 6 for the numbers of cells counted in the RGC layer of the normotensive eyes of both treatment groups.

Table 1.

 

Normalized OD RGC Counts from Animals with Moderate IOP Elevation in the Two Treatment Groups

Monkey #	Inferior Cell Counts (Sample 2,6,7) OD/OS	Superior Cell Counts (Sample 1,4,5) OD/OS	Peripheral Cell Counts (Sample 4,5,6,7) OD/OS	Perimacula Cell Counts (Sample 1,2,3) OD/OS	Perifoveal Cell Counts (pF) OD/OS	Total Cell Counts (Sample 1–7,pF) OD/OS	IOP̄ (mm Hg)
Vehicle
96	0.83	0.87	0.91	0.71	0.92	0.87	37.4
98	0.78	0.77	0.77	0.84	0.96	0.90	31.2
106	0.69	0.92	0.73	0.96	0.69	0.74	28.9
105	0.84	1.04	0.79	0.92	1.08	1.02	27.3
Average	0.78	0.90	0.80	0.86	0.91	0.88	31.2
SEM	0.04	0.06	0.04	0.06	0.08	0.06	2.20
Memantine
94	0.97	1.16	0.89	1.12	0.63	0.78	35.3
97	0.88	0.74	0.71	0.82	0.91	0.86	29.3
107	0.89	0.85	0.92	0.84	0.88	0.89	29.2
99	1.00	1.00	0.97	0.88	0.91	0.89	26.2
Average	0.94	0.94	0.87	0.91	0.83	0.86	30.0
SEM	0.03	0.09	0.06	0.07	0.07	0.03	1.9
P-value*	0.01	0.69	0.36	0.66	0.49	0.69	0.15

*  Comparison of two treatment groups: two-tailed student t-test.

Figure 6.

 

Summary of RGC counts obtained from the normotensive (OS) eyes of both treatment groups. Results are shown for average counts (± SEM) from each individual retinal sample region as well as total counts summed from all eight retinal sample regions for each treatment group. PF counts and total counts are indicated by the right-hand scale while counts for all other retinal sample regions (1 → 7) are indicated by the left-hand scale. The P values (two-tailed t-test) for a comparison of counts from the two treatment groups are as follows: sample 1 (0.18), sample 2 (0.35), sample 3 (0.45), sample 4 (0.19), sample 5 (0.44), sample 6 (0.19), sample 7 (0.48), sample PF (0.21), Total (0.56). There is no evidence for a toxic effect of memantine treatment on RGC counts in normotensive eyes. On average, a total of over 15,000 cells were counted in the normotensive eyes of each treatment group. See Methods for details of cell counting.

Table 2.

 

IOP Peak and Mean Values for Individual Animals of Both Treatment Groups

Monkey #	Baseline IOP (mm Hg)	Peak IOP (mm Hg)	IOP̄ (mm Hg)
						t1	t2	t3
Vehicle-treated animals
96	22.1 (22.8)	50.0	38.7 (21.5)	39.9 (19.5)	38.4 (19.5)
104	19.1 (19.5)	61.0	54.5 (23.5)	46.1 (21.0)	39.6 (19.5)
91	18.8 (18.7)	55.0	51.0 (19.0)	51.0 (18.5)	49.6 (20.0)
93	19.0 (19.0)	55.9	53.4 (16.5)	52.2 (19.5)	53.0 (20.5)
92	18.7 (19.1)	61.5	51.1 (18.0)	53.3 (20.5)	54.7 (20.0)
Range	18.7–22.1	50.0–61.5	38.7–54.5	39.9–53.3	38.4–54.7
	(18.7–22.8)		(16.5–23.5)	(18.5–21.0)	(19.5–20.5)
X̄ ± SEM	19.5 ± 0.64	58.7 ± 2.1	49.8 ± 2.8	48.5 ± 2.5	47.0 ± 3.4
	(19.8 ± 0.76)		(19.7 ± 1.2)	(19.8 ± 0.4)	(19.9 ± 0.2)
Memantine-treated animals
102	19.3 (18.7)	55.0	32.7 (16.0)	28.1 (18.0)	26.5 (15.5)
97	21.1 (21.0)	47.0	43.3 (17.0)	37.7 (18.5)	34.3 (19.5)
94	18.5 (18.5)	53.0	43.6 (18.0)	37.5 (17.5)	36.5 (20.0)
95	21.1 (14.7)	60.0	56.9 (21.5)	51.6 (21.0)	45.0 (23.5)
101	20.8 (21.2)	61.5	54.8 (18.5)	58.0 (19.5)	56.6 (19.5)
Range	18.5–21.1	47.0–61.5	32.7–56.9	28.1–58.0	26.5–56.6
	(14.7–21.2)		(16.0–21.5)	(17.5–21.0)	(15.5–23.5)
X̄ ± SEM	20.1 ± 0.53	55.3 ± 2.6	46.3 ± 4.3	42.6 ± 5.3	39.8 ± 5.1
	(18.8 ± 1.1)		(17.8 ± 1.0)	(18.9 ± 0.6)	(18.2 ± 0.2)

 Values for contralateral normotensive (OS) eyes are shown in parentheses.

Table 3.

 

Summary for Linear Regression Analysis of Optic Nerve Head Tomography Measures

		Memantine			Vehicle			P Value, **
		Slope*	r *	Slope*	r *
Cup measures
Mean cup depth	t1	0.050	0.85	0.206	0.61	0.282
	t2	0.033	0.94	0.356	0.85	0.018
	t3	0.008	0.06	0.250	0.94	0.057
Cup volume below surface	t1	0.067	0.72	0.438	0.54	0.314
	t2	0.038	0.87	0.756	0.74	0.059
	t3	0.029	0.62	0.524	0.86	0.019
CDR	t1	0.012	0.66	0.023	0.59	0.580
	t2	0.008	0.52	0.033	0.77	0.217
	t3	0.003	0.19	0.029	0.83	0.116
Cup area	t1	0.055	0.98	0.305	0.56	0.294
	t2	0.071	0.90	0.597	0.84	0.023
	t3	0.072	0.75	0.578	0.94	0.004
Cup shape	t1	−0.073	0.98	−0.101	0.95	0.201
	t2	−0.052	0.98	−0.147	0.90	0.029
	t3	−0.050	0.98	−0.076	0.89	0.261
Neuroretinal rim measures
Rim area	t1	−0.040	0.99	−0.041	0.88	0.955
	t2	−0.031	0.99	−0.049	0.99	0.010
	t3	−0.033	0.95	−0.059	0.95	0.085
Rim/disc	t1	−0.031	0.96	−0.039	0.87	0.554
	t2	−0.024	0.99	−0.048	0.98	0.006
	t3	−0.026	0.94	−0.059	0.94	0.039
Rim volume	t1	−0.057	0.94	−0.037	0.90	0.328
	t2	−0.035	0.92	−0.031	0.97	0.800
	t3	−0.032	0.93	−0.039	0.95	0.553
RNFL thickness	t1	−0.035	0.92	−0.024	0.50	0.637
	t2	−0.017	0.68	−0.042	0.64	0.409
	t3	−0.029	0.84	−0.060	0.94	0.125
RNFL cross section	t1	−0.042	0.91	−0.024	0.50	0.497
	t2	−0.021	0.74	−0.039	0.60	0.564
	t3	−0.033	0.84	−0.062	0.93	0.195

*  Determined from linear regression analysis.

**  Determined from analysis of covariance.

 Values represent measures from all five animals in each treatment group for which HRT measures were made.

Figure 7.

 

Summary of normalized (OD/OS) cup measurements from confocal laser scans at t2 for the five animals having the highest mean IOPs in each treatment group (see Table 2 ). These five animals in each group were the only animals for which scans were made. Values are plotted as a function of mean IOP for measurements of (A) mean cup depth, (B) cup volume below surface, (C) cup area, (D) cup shape (third moment), and (E) global cup/disc ratio. In each panel, linear regression is plotted as a heavy line for vehicle-treated animals (filled squares) or a light line for memantine-treated animals (open circles). See Table 3 for a summary of measurements made at all three time points.

Figure 8.

 

Summary of normalized (OD/OS) neuroretinal rim measurements at t2 including (A) rim area, (B) rim/disc ratio, (C) rim volume, (D) retinal nerve fiber layer (RNFL) thickness, and (E) RNFL cross-section. These measurements were obtained from the same animals (and scans) represented in Figure 7 . Values are plotted as a function of mean IOP. See Table 3 for a summary of measurements made at all three time points.

Figure 9.

 

Plot of three of the five cup measurements shown in Figure 7 from the hypertensive eye of all five animals in each treatment group, without normalizing, at the three measurement time points. Plots of cup volume below the surface in mm³ (A), cup shape (B), and cup area/disc area ratio (C) are shown. The left-hand panels summarize measurements from all five vehicle-treated animals while measurements from the memantine-treated animals are shown in the right-hand panels. Individual animals are represented by symbols and colors as indicated in the top panels.

Figure 10.

 

Plot of three of the five neuroretinal rim measurements shown in Figure 8 from the hypertensive eye of all five animals in each treatment group, without normalizing, at all three measurement time points. Plots of rim area in mm² (A), rim volume in mm³ (B), and retinal nerve fiber layer (RNFL) thickness in mm (C) are included. In each case, results from vehicle-treated animals are plotted in the left-hand panels and results from memantine-treated animals are plotted in the right-hand panels. At all three time points, for all three measurements, the average value for the memantine-treated animals is greater than that for the vehicle-treated animals. This difference is significant (P < 0.05; Student’s two-tailed t-test) for only the RNFL Thickness measure at t1 (indicated by asterisk*).

The authors thank Kuankuan Chen and Jennifer Lee for expert assistance with statistical analysis, and James Burke who performed the laser treatment on all animals used in this study.