The results of clinical
14,17,28 and animal studies
15,16,29 have shown that EEPs originate from stimulated neurons located proximal to the photoreceptors. In addition, the function of the optic nerve and transmission of the signals by the optic nerve axons are necessary for the EEPs to be generated. A transection of the optic nerve or an intravitreal injection of tetrodotoxin has been shown to abolish the EEPs.
16,29
On the other hand, the threshold for eliciting phosphenes by TES is elevated significantly in eyes with RP.
10,11,18 Several studies have shown that 5 to 10 times stronger currents were needed to evoke a phosphene by TES in RP patients than in the controls.
10,18 The reason for this has not been definitively determined but a degeneration of the RGCs has been suspected
18 because optic atrophy is one of features of the fundus of RP patients. In addition, some histologic studies have confirmed a reduction in the number of RGCs in the retina and axons in the optic nerve of eyes of RP patients.
27,30–32
We have successfully recorded EEPs from WT rabbits. The waveforms of WT rabbits had two positive peaks, P1 and P2, that were elicited by 2 to 5 mA of electrical current. These values are comparable to those of earlier studies using normal albino rabbits.
20,29 In addition, EEPs have been elicited by TES of rabbit retinas with severely degenerated photoreceptors, as reported.
21,33 The amplitudes of EEPs were reduced and thresholds for eliciting EEPs were significantly elevated in the Tg rabbits with highly degenerated retinas. However, the implicit times of P1 in Tg rabbits were not delayed. These results resemble those from RP patients in which the EEPs were elicited by TES.
13 Our data suggest that we need to evaluate the residual visual pathway in RP patients by the amplitudes of the P1 rather than by the implicit times. However, there still is a possibility that the arrival of the neural signals in Tg rabbits were being masked rather than not delayed. In Tg rabbits, various electrical potentials might be generated by retinal remodeling,
8,9 and these potentials could contribute to the masking of the P1 component by overlapping and/or subtracting between P1 components and potentials generated by retinal remodeling, which might result in normally apparent implicit times.
The decrease in the number of axons in the optic nerve of Tg rabbits was also comparable to the findings in morphometric studies of postmortem eyes of RP patients
27 and some rodent models of RP.
34,35
Our results showed a discordance between the extent of reduction in the axon numbers and the reduction of the EEPs; the EEPs were severely reduced even in Tg rabbits that had a similar number of axons as the WT rabbits, and the ratio of the mean amplitude of the EEPs to the number of axons (EEP amplitudes/number of axons) was significantly decreased in Tg rabbits. These results indicated that the decreased amplitude of the EEPs could not be simply explained by the reduction in the number of axons in the optic nerve.
There are several possible explanations for this discordance between the amplitude of the EEPs and the number of axons in Tg rabbits. First, the number of counted axons in the optic nerve may be preserved but their ability to transmit the neural signals may have been damaged. As shown in
Figures 3F and
3I, the optic nerves in Tg rabbits appeared to include some degenerated axons, although an accurate differentiation between healthy and degenerative axons is difficult. Our axon counting technique probably included a number of degenerated axons; therefore, the EEPs were reduced despite the apparent preservation of the number of axons in Tg rabbits. To confirm this possibility, an evaluation of optic nerve function will be necessary.
A second possible reason is that the amplitudes of the EEPs were altered by not only the axon numbers but also the physiology of the inner retinal layers. In Tg rabbits, extensive functional remodeling of the second- and third-order neurons has been reported during the course of retinal degeneration.
36,37 Histologic studies have revealed a reprogramming of inner nuclear cells at a relatively early stage of degeneration,
38 and an extensive gliosis develops throughout the retina at the end stage of retinal degeneration in Tg rabbits.
39 This inner retinal disorganization might contribute to the reduction in the amplitude of the EEPs in Tg rabbits. An earlier study reported that responses of the on-type (ON)-bipolar cells to TES were significantly reduced after an intravitreal injection of 2-amino-4-phosphonobutyric acid, which blocks ON-bipolar cells selectively.
15 This supports our second possibility because this earlier study suggested that the ON-bipolar cells and their related synaptic sites, which are located in the inner retinal layers, were also involved in the generation of the EEPs.
A third possibility is that the neural signals in the lateral geniculate nucleus, optic radiation, and the visual cortex have degenerated by the photoreceptor degeneration. Several studies have reported a degeneration of the optic radiation in RP patients,
40,41 although the precise mechanisms have not been determined. Remodeling of the cortex could potentially also occur due to a loss of functional input rather than axon degeneration, which an optic nerve axon count would not detect. But, if damages in the postoptic nerve signal were caused by transsynaptic degeneration from the retina, contribution of this pathway might not be so large because the numbers of the optic nerves were relatively preserved compared to the EEPs in our Tg rabbits.
We suggest that all three of these mechanisms might have contributed to the reduction of the EEPs to a greater or lesser degree. If our second hypothesis is correct, we need to be cautious in selecting suitable candidates for an electronic retinal implant in the end stage of RP patients. The thresholds for eliciting phosphenes by TES are elevated in many end-stage RP patients, but some of them might have a considerable number of axons. Thus, they are still good candidates for the implantation of retinal prosthesis, especially in an epiretinal array with electrical stimulation, which directly stimulates the RGCs.
There are some limitations in our study. The total number of axons can be highly dependent on the counting technique used and the individuals doing the counts. A large variation in axon counts of normal optic nerves is reported even when controlling for age.
42,43 In the current study, we fixed the optic nerve soon after euthanasia and, thus, reduced the tissue damage caused by a delay in fixation, which is a problem in morphometric studies of postmortem human eyes.
The second limitation is that we did not perform quantitative analysis of the degenerated retina, that is, we did not count the number of cells, including the retinal bipolar cells and the RGCs. We confirmed the retinal section of one Tg rabbit, which had a severe degeneration of the photoreceptors. A more accurate analysis of the inner retinal morphology might be helpful in determining the mechanisms causing the reduction of the EEPs in Tg rabbits.