Electrically Evoked Potentials Are Reduced Compared to Axon Numbers in Rhodopsin P347L Transgenic Rabbits With Severe Photoreceptor Degeneration

Abstract

Purpose: To determine the relationship between the amplitudes of the electrically evoked potentials (EEPs) and the number of optic nerve axons at a late stage of retinal degeneration in rhodopsin P347L transgenic (Tg) rabbits, a model of retinitis pigmentosa.

Methods: Six eyes of six wild-type (WT) (43.8 ± 7.5 months of age) and six eyes of six Tg (40.3 ± 2.6 months of age) rabbits were studied. The EEPs were elicited by 1 to 5 mA of transcorneal electrical stimulation. The first positive wave, the P1 component, was analyzed. After euthanasia, the number of axons in the optic nerve was counted.

Results: The threshold current to elicit a P1 was significantly higher in Tg rabbits than WT rabbits. The amplitude of P1 elicited by 5 mA in Tg rabbits was about 24% of that in WT rabbits (P < 0.01). The number of axons in the optic nerve of Tg rabbits was reduced to about 59% of that of WT rabbits (P < 0.01). The correlation between the axon number and the amplitude of the P1 in Tg and WT rabbits was not significant. The mean ratio of the P1 amplitude/axon in Tg rabbits was decreased to 53% of that in WT rabbits (P < 0.05).

Conclusions: The degree of reduction in the EEP in Tg rabbits is more severe than the reduction in the number of optic nerve axons. The use of transcorneal electrical stimulation to determine the suitable candidates for prosthesis at the end-stage of retinitis pigmentosa may underestimate the condition of the optic nerves.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal disorders characterized by a progressive degeneration of the photoreceptors leading to a reduction of vision. The major symptoms of RP are impaired night vision, slow progressive peripheral-to-central visual field loss and eventually blindness. To try to restore vision in these advanced RP patients, retinal prostheses have been used to stimulate the residual functioning retinal neurons by electrical currents.^1–3 These prostheses consist of an array of electrodes that are implanted on⁴ or beneath the retina⁵ or in the suprachoroid space.⁶ They are used to deliver electrical currents to activate the residual functioning retinal neurons to send signals to the visual cortex. 

The outcomes of the prosthesis systems differ significantly, ranging from “no benefit” to the ability to read large letters with a measurable visual acuity.^1,2,7 The degree of success of these methods is suspected to be dependent on the functionality of the inner retinal neurons and optic nerve because it is known that photoreceptor degeneration leads to a gradual deconstruction and functional reprogramming of the middle and inner retina or remodeling.^8,9 Thus, determining the functionality of the residual visual pathway is needed before performing an invasive surgical procedure of implanting a prosthesis in eyes with advanced RP. 

Electroretinograms (ERGs) and visual evoked potentials are useful methods to evaluate the retinal function and the integrity of the visual pathways. However, these tests cannot assess RP patients at advanced stages because the photoreceptors are degenerated. In these cases, transcorneal electrical stimulation (TES) has been used to determine the functionality of the residual second- and high-order neurons in RP patients at advanced stages.^10,11 The threshold for evoking a light sensation, called a phosphene, is a subjective way to analyze the functionality of the inner retinal neurons. On the other hand, the electrically evoked potentials (EEPs) recorded from the visual cortex and elicited by TES are an objective method to evaluate the integrity of the afferent visual pathways, including the inner retina and optic nerve to the visual cortex.^12–14 In addition, investigations of the EEPs have enabled researchers to evaluate the residual retinal functions objectively, even in animals.^15,16 The type of retinal neurons activated by TES has not been definitively determined, but reductions of the amplitude of the EEPs in patients with optic atrophy¹⁷ and abolishment of the EEPs after optic nerve transection in animals suggested a need of functioning retinal ganglion cells (RGCs). 

In eyes with RP, there is a marked reduction of the amplitudes of the EEPs or significant elevations of the thresholds for eliciting EEPs or phosphenes by TES.^10,11,13,18 To date, these changes were suspected to be related to the degeneration of the RGCs due to retinal remodeling^8,9 following the photoreceptor degeneration. However, the exact mechanisms causing these alterations have not been determined. 

The purpose of this study was to determine the relationship between amplitudes of the EEPs and the number of axons in the optic nerve in the rhodopsin Pro347Leu transgenic rabbit (Tg), a model of RP.¹⁹ 

Materials and Methods

Animals

All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines for the Use of Animals of the Nagoya University Graduate School of Medicine. The Nagoya University Animal Experiment Committee approved this study (approval number 30107). The creation of this Tg rabbit has been described in detail.¹⁹ The rabbits were maintained on a 12-hour light:12-hour dark cycle with an ambient illumination of <40 lux. 

Older Tg rabbits were selected because ERG studies have shown that the ERGs were essentially absent in these older rabbits. Before beginning the experiments, we confirmed that the amplitudes of the full-field ERGs elicited by flash stimuli of 2.5 log cd-s/m² from Tg rabbits were <10 μV under both scotopic and photopic conditions. Six eyes of six wild-type (WT) rabbits whose mean ± standard deviation (SD) age was 43.8 ± 7.5 months and six eyes of six Tg rabbits whose mean ± SD age was 40.3 ± 2.6 months were used. Both types of the rabbits were the New Zealand White strain. None of the eyes had been treated with any intravitreal injections or ocular surgeries. The rabbits were anesthetized with an intramuscular injection of 25 mg/kg ketamine and 2 mg/kg xylazine with topical oxybuprocaine for the cornea, and the pupils were fully dilated with topical 0.5% tropicamide and 0.5% phenylephrine HCl. 

Fundus Photographs

Color photographs of the fundus (CX-1; Canon Inc., Tokyo, Japan) of one eye of a 52-month-old Tg rabbit and one eye of a 44-month-old WT rabbit were taken. These eyes were also used for analyses of the EEPs. 

Electrically Evoked Potentials

We recorded EEPs from one eye of six WT and six Tg rabbits, and the techniques have been described in detail.^20,21 Briefly, we removed the skin covering the skull and drilled 1-mm holes through the skull at 7 mm to the right of the midline for the right visual cortex and 7 mm left of the midline for the left visual cortex. The holes were located 8 mm anterior to the lambda suture. Another hole was drilled at the intersection of the bregma and the midline for the reference electrode. Next, silver-coated stainless steel screws were screwed into the skull to make electrical contact with the dura mater. They served as the pick-up electrodes. 

Electrical stimuli were delivered by a bipolar contact lens electrode (Gold Lens; Doran Instruments, Littleton, MA, USA). Hydroxyethyl cellulose was used to keep the cornea and conjunctiva hydrated and to ensure good electrical contact between the electrodes and the cornea and conjunctiva. 

The EEPs were recorded from the right visual cortex when the TES was applied to the left eye and recorded from the left visual cortex when the TES was applied to the right eye. The ground electrode was placed on the ear. An electronic stimulator with a stimulus isolation unit (BSI-2; Bak Electronics, Inc., Umatilla, FL, USA) and a biphasic signal generator (RP-1; Bak Electronics, Inc.) were connected to the bipolar contact lens electrode. 

Five hundred responses were averaged using a computer-assisted signal averaging system (Neuropak S1; Nihon Kohden, Tokyo, Japan). Biphasic electrical pulses with currents from 1 to 5 mA were used to elicit the EEPs. The pulses were square waves of 0.5-millisecond duration.²¹ We recorded two sets of EEPs for each rabbit in one session, and the EEPs with the larger amplitudes were used for the analyses. 

The P1 component of the EEP is the first positive wave with an implicit time of about 30 milliseconds after stimulation. The N1 and P2 components were the negative wave and positive wave that were evoked after the P1. The threshold current to elicit the P1 component and the amplitudes of P1 components were analyzed because these values were reported to be useful indicators of the EEPs and were also less influenced by general anesthesia in earlier studies.²⁰ 

The amplitude of P1 was measured from the baseline, that is the electrical potential at stimulus onset, to the positive peak of P1, and the implicit time of P1 was measured from stimulus onset to the peak of P1. The thresholds of the electrical current to elicit P1 were used for the statistical analyses. The measurements of the amplitude of the P1 component were made by a masked examiner (AK). 

Optic Nerve Tissues and Retinal Sections

The optic nerves of the six eyes of six Tg rabbits and six eyes of six WT rabbits and retinal sections of the one eye of one Tg rabbit and one eye of one WT rabbit were evaluated histologically after recording the EEPs. Soon after the rabbits were euthanized, the eyes were enucleated with caution to preserve a segment of the optic nerve as long as possible. The retinas were used for histologic analyses, as previously described in detail.²² The optic nerves were approximately 1 to 5 mm posterior to the sclera. They were fixed in 1% osmium tetroxide in phosphate-buffered saline. The nerves were processed and embedded in epoxy resin and semithin sections (1 μm) were cut perpendicular to the long axis of the optic nerve. The sections were stained with 1% toluidine blue and observed by light microscopy (BX61 microscope with digital photograph system DP70-BSW; Olympus. Tokyo, Japan). 

Counting Numbers of Optic Nerve Axons

The number of axons was counted from the sections stained with toluidine blue. First, the optic neural area was measured in the low power images of the optic disc (6×), and the optic sheath, connective tissue, and vessels were removed from the entire area of the optic nerve by using the ImageJ software (version 1.48; http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).²³ To examine the optic nerve tissue in more detail, we ran the command “Type” → “8-Bit” → “edit” → “clear outside” to exclude the tissues outside the bundles of axons, → “adjust” → “threshold (default)” to show only the actual axons as much as possible, and → “analyze the particles (particles ranged from 10 to 10,000,000 pixels at a circularity range 0 to 1.” The areas representing the actual axons were chosen to combine by using the “roi manager” attached to the ImageJ software, and we measured these combined areas as the total area. 

Next, we ran the command “invert” → “to analyze the particles (particles ranged from 50 to 5000 pixels at circularity range of 0 to 1)” to obtain the areas other than the axons such as the vascular structures and extracellular space. We combined these areas by using the roi manager and measured the area (the other area). 

We determined the axon area as the area determined by subtracting the other areas from the total area. 

Second, we selected three rectangle areas of 800 × 600 pixels (272 × 204 μm), which contained fewer vessels and less connective tissue, located in a different quadrant, from the high power images (60×). We counted the number of myelinated axons in these areas by running the command “Type” → “8-Bit” → “Process” → “Find Maxima…at a noise tolerance of 10 and the output style was segmented particles)” → “analyze particles (range from 0 to 30 μm² at circularity 0 to 1)” by referring to the semiautomated method.²⁴ We considered the particles chosen by these processes as those indicating actual axons. The density of axons in each selected square region was calculated by dividing the number of the axons by its size (0–5, 5–10, 10–15, 15–20, 20–25, and 25–30 μm²) in the square by the area size (272 × 204 μm²). The mean axon density from three selected regions was used as the axon density of each eye. We estimated not only the total number of axons but also the number of axons of different diameters. The axon numbers were calculated by multiplying the neural area of entire optic nerve in the cross-section image and mean axon density of each eye. 

Statistical Analyses

Mann-Whitney U tests were used to determine the statistical significance between two groups, and repeated measure ANOVA was used for statistical analyses of the difference in axon numbers between WT and Tg rabbits for multiple comparison depending on axon diameters. Spearman correlation tests were used to determine the significance of the correlations between the P1 amplitude and the number of axon in the optic nerve. A P < 0.05 was considered statistically significant. 

Results

Fundus Appearance and Retinal Histology

A comparison of the fundus image of the WT and Tg rabbit showed that the retinal vessels were narrower in the Tg rabbit than in the WT rabbit (Figs. 1A, 1B). Narrow vessels are one of the features of the retina of RP patients.^25,26 However, no other specific abnormalities of the optic disc and retina were observed in the Tg rabbit. 

Figure 1

 

Fundus photographs and light microscope photomicrographs of retinal sections of (A, C) WT, 44-month-old, and (B, D) Tg, 52-month-old rabbits. (C, D) Scale bar: 50 μm.

Representative retinal sections in the area of the visual streak of the WT rabbit and the Tg rabbit are shown in Figures 1C and 1D, respectively. The photoreceptor layer and the outer nuclear layer were completely absent in the Tg rabbit. In addition, the surface of retina was uneven due to the larger stoma. 

Evaluation of EEPs

Representative intensity-response series of EEPs from the WT rabbit at 56 months of age and those of the Tg rabbit at 39 months of age are shown in Figure 2A. In the WT rabbit, distinct EEPs were elicited by 2 mA of electrical current, and on the other hand, the EEPs became recordable only with 4-mA electrical current in the Tg rabbit. 

Figure 2

 

EEPs from Tg and WT rabbits. (A) Representative intensity response series of EEPs of WT and Tg rabbits. The stimulus intensities ranged from 1 to 5 mA in 5 steps. The P1 component of the EEPs (arrows) was defined as the first positive wave with an implicit time of about 30 milliseconds after the stimulus onset. The N1 and P2 components were defined as the negative wave and positive wave that emerged after P1, respectively. The horizontal broken lines indicate the baseline potential. (B) The EEPs elicited with 5 mA from five other WT and five other Tg rabbits. Comparison of amplitude of P1 (C) and implicit time (D) of P1 stimulated with 5-mA current between WT and Tg rabbits. There is a significant difference between WT and Tg rabbits in the amplitude of P1 (P < 0.01, Mann-Whitney U test) but not in the implicit time. Error bars indicate SDs.

The EEPs from all other WT and Tg rabbits stimulated with 5 mA are shown in Figure 2B. The threshold currents to elicit P1 were significantly higher in Tg rabbits (P < 0.01, Mann-Whitney U test). The threshold current was 4 mA in two Tg rabbits and 5 mA in four Tg rabbits, whereas it was 2 mA in three WT rabbits, 3 mA in two WT rabbits, and 4 mA in one WT rabbit. 

The amplitudes of the P1s in six Tg rabbits elicited by 5 mA were significantly smaller than those in six WT rabbits (Fig. 2C; P < 0.01, Mann-Whitney U test). On the other hand, the implicit times of P1 of EEPs with a 5-mA current in Tg rabbits were not significantly different from those in WT rabbits (Fig. 2D; Mann-Whitney U test). 

Sections of Optic Nerve Tissue

Representative cross-sectional images of the optic nerve of one WT rabbit at 52 months of age and two Tg rabbits at 39 months of age are shown in Figure 3. The optic nerve was composed of a neural area stained in blue and connective tissue area, which lacked the blue staining in the low magnification images. The overall size of the optic nerve of WT rabbit was larger than those of Tg rabbits (Figs. 3A, 3D, 3G). The mean ± SD neural area of six WT rabbits was 1.43 ± 0.26 mm² and that of the six Tg rabbits was 1.13 ± 0.70 mm² (P < 0.01, Mann-Whitney U test). The optic nerves of some Tg rabbits contained more connective tissue between the nerve fascicles, resulting in a decrease in the total neural area (Fig. 3D, Tg 1). However, other nerves had a packed neural area with less connective tissue (Fig. 3G, Tg 4). 

Figure 3

 

Light microscope photomicrographs of optic nerve cut in cross-section and stained with toluidine blue from WT 1 (A–C), Tg 1(D–F), and Tg 4 (G–I). Low (A, D, G), moderate (B, E, H), and high (C, F, I) magnified images are shown. The magnified images of the white squares in A, D, G are shown in B, E, H, respectively. Magnified images of white squares in B, E, H are shown in C, F, I, respectively. The red arrows in F and I point to densely stained myelinated axons. Some of myelin ring cannot be distinguished due to the staining of the entire structure (yellow arrowheads in C, F, I). These arrows and arrowheads represent examples of degenerated axons. The overall size of the optic nerve of WT 1 (A) is larger than those in Tg rabbits (D, G). Optic nerve in Tg 1 (D) was composed of more connective tissue between the nerve fascicles than in the WT 1 (A) and Tg 4 (G). The total numbers of axons were 231,300 for WT 1, 56,169 for Tg 1, and 140,097 for Tg 4 rabbits. (A, D, G) Scale bar: 100 μm. (B, E, H) Scale bar: 25 μm. (I, C, F) Scale bar: 5 μm.

Magnifying the neural area made the blue-stained myelinated axons visible. In both WT and Tg rabbits, the optic nerve axons were clearly visible in the high-magnified images. However, more myelinated axons were densely stained (Figs. 3F, 3I, red arrows) and a higher number of myelin rings could not be distinguished due to staining of the entire structure in Tg rabbits compared to WT rabbits (Figs. 3C, 3F, 3I, arrowheads). These findings were most likely due to the degenerated axons,²⁷ which might have contributed to the overestimation of the number of axons. 

Evaluation of Axons in Optic Nerve

The different sizes (in 5-μm² steps) of the optic nerve axons in six WT rabbits and in six Tg rabbits are shown in Figure 4. The total number of axons in Tg rabbits was significantly lower than that in WT rabbits (P < 0.01, Mann-Whitney U test). The mean ± SD total number of axons in Tg rabbits was 109,049 ± 50,661, which was about 59% of that in WT rabbits at 185,167 ± 38,299. The axon numbers for each axon size of WT rabbits had a tendency to be larger than those of Tg rabbits, although there were significant interactions between the effects of axon size and rabbits (WT and Tg rabbits; repeated measure ANOVA). 

Figure 4

 

Distribution of the diameter of optic nerve axons from WT (n = 6: black) and Tg (n = 6: red) rabbits calculated from the cross-sectional images of the optic nerves. The total axon number in Tg rabbits was significantly fewer than that in WT rabbits (P < 0.01, Mann-Whitney U test). Error bars indicate SDs.

Correlations Between P1 Amplitude and Number of Optic Nerve Axons

The P1 amplitudes of the EEPs and total axon numbers of each rabbit are plotted in Figure 5A. The correlations between axon number and amplitude of P1 in Tg rabbits and WT rabbits were not significant (Spearman correlation tests). The amplitudes of P1 in all Tg rabbits were less than 20 μV and those in the WT rabbits were all larger than 20 μV. However, the number of axons in two Tg rabbits (numbers 2 and 4) were comparable to the lowest number of axons in the WT rabbits. The amplitudes of P1 were reduced even in these Tg rabbits that had equal number of axons as WT rabbits. These findings indicate that the preserved axons in Tg rabbits are not related solely to preservation of the amplitude of the P1. 

Figure 5

 

(A) Relationship between the amplitude of P1 of the EEP and the number of axons in optic nerve. The plots of WT rabbits are shown in black and those of Tg rabbits are shown in red. No significant correlations were found between the amplitude of P1 and the number of axons in either WT or Tg rabbits (Spearman correlation test). (B) Comparisons of ratio of P1 amplitude per axon in WT rabbits and Tg rabbits. The average ratio was significantly smaller in Tg rabbits than that in WT rabbits (P < 0.05, Mann-Whitney U test). These data indicate each axon of Tg rabbits could not transmit the signal to the visual cortex as efficiently as WT rabbits did. Error bars indicate SDs.

To examine this relationship in more detail, we calculated the P1/axon number ratio. A comparison of the ratio in the WT and Tg rabbits is shown in Figure 5B. The average ratio was significantly smaller in Tg rabbits than that in WT rabbits (P < 0.05, Mann-Whitney U test), which indicates that the afferent visual pathway from the inner retina to the visual cortex did not transmit the signals as effectively as the WT rabbits did in spite of the preserved number of the axons in Tg rabbits. 

Discussion

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 suspected¹⁸ 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 patients²⁷ 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. 

Conclusions

Our results showed that both the amplitudes of the EEPs and the numbers of axons in the optic nerve of older Tg rabbits were reduced, but the degree of reduction in the EEPs was more severe than the reduction in the number of axons. In Tg rabbits, the reduction in the amplitude of the EEPs might not be related solely to the number of axons in the optic nerve but also to the gliosis of the inner retina or the ability to transmit signal in the optic nerve axons. Our data indicate that the results of TES, which is being used to determine suitable candidates for prosthesis in advanced RP patients, might underestimate the physiologic condition of the postphotoreceptor neurons in eyes with RP. 

Acknowledgments

The authors thank Duco Hamasaki of the Bascom Palmer Eye Institute for the discussions and editing the final version of the manuscript. 

Supported in part by Grant-in-Aid for Scientific Research C (number 16K11320 to SU) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.jsps.go.jp/) and by the Takayanagi Retinal Research Grant. 

Disclosure: T. Kominami, None; S. Ueno, None; K. Nishida, None; D. Inooka, None; A. Kominami, None; M. Kondo, None; H. Terasaki, None