In conducting GDx VCC measurements in nonhuman primates, there are several potential sources of variability that should be monitored. Subjects cannot fixate, so the operator has greater responsibility for alignment. Vigilance is required to align images consistently from one session to the next, as with human patients who do not fixate well or vary in the ability to comfortably position their head and neck from visit to visit. Contact lenses, which increase image quality and are useful to maintain corneal hydration, can change refraction, apparent corneal curvature, and axial length values. There was a tendency in the current study for monkeys to become myopic over time in the ExpG eye. Refraction values cannot be assumed to be constant. This change might impact the computation of some values made by the software. Collecting session-specific information will help assess the impact of this potential source of variability. Collecting multiple discrete scans at each time point, especially at baseline, to get a measure of individual eye variability, can reduce confidence intervals, increasing the likelihood of identifying meaningful change. Inter- and intrasession variability reported in the current study were similar to those obtained in an unrelated study of eyes of 10 normal cynomolgus monkeys (unreported data).
Terminal GDx parameters, especially TSNIT, were highly correlated with axon counts and c/d ratio changes. The correlation of individual eye GDx parameter values with the corresponding axon counts, although significant, was not as strong as when the GDx results were first expressed as a percentage change in ExpG compared with Fellow eyes (). This suggests that there are individual animal traits that can cause variation in the GDx measures or in the axon counts. Unlike the Hare study,10
the current study did not detect any memantine-related effects on structural parameters during ExpG. However, only monkeys with the highest mean IOPs were studied by Hare, and the structural measures were done with the Heidelberg Retina Tomograph. The current study also did not detect any significant decrease in optic nerve area, presumably because of the relatively mild loss of axons (<35% loss comparing ExpG to Fellow axon counts) and shorter duration of IOP elevation as compared with other studies.1,28
Evidence for a neuroprotective effect of memantine on ganglion cell function in this study could manifest as either enhanced or stabilized mfVEP from ExpG eyes. The authors found that mfVEP from the central stimulus element was sensitive to ExpG: Animals receiving no memantine exhibited significantly lower mfVEP amplitudes in ExpG eyes compared with baseline in the same eye or the final value in the Fellow eye. In contrast, ExpG eyes from memantine-treated animals had higher overall mean amplitudes that were not significantly different relative to baseline in the same eye and final test in the Fellow eye (). Note that at baseline the mfVEP RMS SNR determinations of the ExpG and Fellow eyes (5.94 ± 0.91 and 6.58 ± 1.00) of the group of animals that were administered memantine tended to be lower (but not significantly) than for the ExpG and Fellow eyes (7.04 ± 0.85 and 7.17 ± 1.23) of the group of animals that did not receive memantine. In addition to inherent variability in mfVEP recordings, memantine plasma levels at the time of sacrifice also were quite variable. Serum levels of memantine were not maintained at the 1 μM level achieved in the Hare studies although early on in their study, levels fluctuated as well.9
Given baseline differences, serum memantine levels, and the variability between animals and test sessions in this study, intra-ocular comparisons rather than between-group comparisons are best suited for evaluating the effects of memantine. As noted, there was a significant interocular difference between the mfVEP of ExpG and Fellow eyes of the no-memantine group; however, there was no statistical difference between ExpG and Fellow eyes in memantine-treated animals. The absence of a difference in mfVEP between ExpG and Fellow eyes in the memantine group is therefore consistent with a stabilization of mfVEP of the ExpG eyes, and thus also consistent with, but not probative of, a possible neuroprotective role of memantine on retinal ganglion cells.
There are several possible reasons why the functional (mfVEP) and structural measures (GDx and axon counts) were not always correlated. Animals with electrophysiologic testing were a smaller subset of the animals with both GDx and axon counts. There are also differences in the variance of the functional and structural measures. The coefficient of variation (SD/mean) for mfVEP RMS SNR is approximately double that of GDx and axon counts. In addition, the relationship between functional and structural measures is not likely to be linear across cell layers or uniform within all animals. Most importantly, the mfVEP measure is derived from the macular stimulus whereas the most severe axonal damage was located in the peripheral optic nerve ().
Despite these limitations, the authors' mfVEP results are similar to the VEP findings of Hare9
in that they suggest that memantine may be providing some neuroprotection of the retinal ganglion cells or other cellular structures subserving the central cortical response. In the Hare studies,9
functional measures demonstrated that memantine treatment delayed but did not prevent the amplitude reduction in the multifocal electroretinogram and visually evoked cortical potential. Amplitude differences were most noticeable at 3 to 5 months after IOP elevation. For monkeys with moderate IOP elevation, memantine treatment was associated with enhanced survival of retinal ganglion cells, especially in the inferior retina. However, they were uncertain if memantine was enhancing the electrophysiology response versus protecting retinal ganglion cells and their axons. It is possible that memantine treatment enhances the ability of surviving retinal ganglion cells to drive activity in the visual cortex, that is, memantine treatment may be associated with plastic changes occurring at more central levels of the visual pathway. Indeed, there was significantly less neuron shrinkage in the lateral geniculate nucleus (LGN) of the memantine-treated than in the vehicle-treated control group even though there was no difference in optic nerve fiber loss.29
LGN neurons showed reduced dendrite complexity and length that were modified by NMDA receptor blockade with memantine.30
As shown by Harwerth,4
electrophysiologic methods can be as sensitive as standard automated perimetry (SAP) in assessing neural loss from ExpG in nonhuman primates. Visual sensitivity losses were not correlated with retinal ganglion cell losses until a substantial number of neurons were lost. There is a period of progressive neural loss that cannot be detected by SAP as is the case with human glaucoma.31
However, objective measures such as the photopic negative response, which represents the linear sum of signals from all of the retinal ganglion cells, might detect a proportional decrease in neuronal loss.4
At any given point of glaucoma progression, it is speculated that the total retinal ganglion cell population includes a mixed population of normal, dysfunctional, nonviable, and atrophic retinal ganglion cells in various stages of degeneration. Current clinical imaging technologies are capable only of quantifying structural changes in retinal ganglion cell axons but do not specify the level of retinal ganglion cell dysfunction.32
Several imaging technologies are now available that are useful for detecting progression during early stages of the glaucoma before functional changes can be detected.7,33–35
Our studies confirm that GDx VCC can be used in monkey ExpG studies to detect early retinal structural changes that may or may not lead to central functional changes. Although the authors' studies show a strong correlation between GDx VCC parameters and axon loss in ExpG, this correlation may not always hold if acute axonal injury affecting birefringence precedes structural loss. Fortune et al. found that a decline in GDx parameters preceded retinal nerve fiber layer (RNFL) thickness changes in monkeys after optic nerve transection36
and after intravitreal colchicine injection.37
Thus, measures of birefringence may provide data on RNFL disease that are complementary to other imaging modalities.