N-Methyl-N-Nitrosourea–Induced Acute Alteration of Retinal Function and Morphology in Monkeys

Abstract

Purpose: The purpose of this study was to investigate both functional and morphologic alteration of the retina acutely induced by N-methyl-N-nitrosourea (MNU) in monkeys.

Methods: The MNU was administered intravenously at a single dose of 40 mg/kg to six cynomolgus monkeys, and standard full-field electroretinograms (ERGs) were recorded 1, 3, and 7 days after dosing. In addition, the rod and cone a-waves in response to high-intensity flashes were analyzed by the a-wave fitting model (a-wave analysis). The photopic negative response (PhNR) was also recorded at the same time points. Furthermore, the retinas of two animals each were examined histopathologically 1, 3, or 7 days after dosing.

Results: The MNU attenuated all the standard full-field ERGs including the rod-driven and cone-driven responses; in the combined rod-cone response, the b-wave was more affected than the a-wave. In the a-wave analysis, the sensitivity parameters (S) of the rod and cone a-waves had decreased on the day after dosing and remained unchanged thereafter. The maximum response parameter (R_max) of the rod a-wave gradually decreased. On the other hand, the R_max in the cone a-wave transiently increased on the day after dosing and decreased thereafter; the PhNR amplitude showed a similar time course change. Histopathologically, the retinal lesion on the day after dosing mainly consisted of pyknosis and karyorrhexis in the photoreceptor nucleus. Depletion of some photoreceptor nuclei, and shortening and disorientation of the photoreceptor segments became prominent at 3 and 7 days after dosing. Localization of degenerated photoreceptors was consistent with that of rhodopsin-positive photoreceptors, resulting in a well-preserved central fovea.

Conclusions: Our results indicated that MNU acutely induced rod-dominant photoreceptor degeneration in monkey retinas, but the photoreceptor function was impaired in both the rods and cones. Functional involvement of the postreceptoral components was also indicated.

N-methyl-N-nitrosourea (MNU), an alkylating agent that interacts with DNA, specifically targets retinal photoreceptor cells, causing acute retinal degeneration via apoptosis in several animal species.¹ Thus, MNU-induced retinal degeneration in experimental animals has been investigated to elucidate the mechanisms of photoreceptor apoptosis.^2–5 This pathologic condition could also be an animal model for photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). 

The majority of reported studies with MNU-induced retinal degeneration in animals, however, have been done in rodents,^2,6–9 species in which the densities of the rod and cone photoreceptors within the retina differ considerably from those in humans. This difference between model animals and humans is a limitation when results in animals are extrapolated to humans. Tsubura et al.¹⁰ reported MNU-induced retinal change in monkeys, a species in which the anatomical structure of the retina is similar to that in humans. They histopathologically examined the retinas from MNU-treated Japanese monkeys (Macaca fuscata), but functional assessment was not performed. Establishment of an animal model highly relevant to the clinical setting is expected especially for testing therapeutic interventions including surgical procedures. 

Therefore, we investigated MNU-induced retinal alterations both functionally and morphologically in monkeys. To characterize the retinal lesions, we serially assessed the electrophysiology and histopathology of the retina after a single intravenous administration of MNU to monkeys. 

Methods

Animals

A total of eight cynomolgus monkeys (Macaca fascicularis) of 5 years of age were used in this study. The animals were housed individually in stainless steel cages (width, 60 cm × depth, 68 cm × height, 75 cm) in an animal study room where the environmental condition was set as follows: room temperature, 24°C; relative humidity, 60%; illumination, 12-hour lighting (7 AM to 7 PM) at 300 luces. The animals were fed 100 g/animal/day of pellet food for monkeys (PS; Oriental Yeast Co., Ltd., Tokyo, Japan). Tap water from a feed-water nozzle was supplied ad libitum to the animals. All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Daiichi Sankyo Co. Ltd. 

Study Design

Two and six animals were randomly assigned to the vehicle- and MNU-treated groups, respectively, as shown in Table 1. In these animals, a series of electroretinograms (ERGs) was recorded 15 and 7 days before dosing and 1, 3, and 7 days after dosing as described below. In addition, two animals each in the MNU-treated group were necropsied 1, 3, or 7 days after dosing. One vehicle-treated animal was also necropsied 7 days after dosing. 

Table 1

 

Group Allocation and Clinical Findings

 

Drug Administration

The MNU (Sigma-Aldrich Corp., St. Louis, MO, USA) was dissolved at a concentration of 8.0 mg/mL in physiologic saline (Otsuka normal saline; Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) containing 0.05% acetic acid, followed by filter sterilization. Immediately after preparation, the dose formulation was administered intravenously via the saphenous vein over 40 minutes to the animals at dose levels of 0 and 40 mg/kg. The dose of 40 mg/kg was set in reference to the previous report,¹⁰ in which a Japanese monkey (Macaca fuscata) treated intravenously with MNU at this level survived until 7 days after dosing and had histopathologic lesions in the retina. 

Clinical Observation

On the day of dosing, clinical signs were observed continuously during intravenous dosing and once each at 1 and 4 hours after the completion of dosing. On the other days, clinical signs were observed once a day in the morning up to the day of necropsy. 

Animal Preparation for ERG Recording

The animals were anesthetized with intramuscular injection of ketamine hydrochloride (Ketalar Intramuscular 500 mg; Daiichi Sankyo Co., Ltd., Tokyo, Japan) (10 mg/kg initial dose, 5–10 mg/kg/h maintenance dose) and 0.6 mg/kg xylazine hydrochloride (Celactal; Bayer Medical Ltd., Osaka, Japan). The pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P ophthalmic solution; Santen Pharmaceutical Co., Ltd., Osaka, Japan); the corneas were anesthetized with topical 0.4% oxybuprocaine hydrochloride (Benoxil ophthalmic solution 0.4%; Santen Pharmaceutical Co., Ltd.) and protected with topical hydroxyethylcellulose (Scopisol solution for eye; Takeda Chemical Industries, Ltd., Osaka, Japan). Prior to ERG recording, the pupil diameter of the left eye was measured to calculate retinal illuminance (in trolands [Td]) for the a-wave analysis. 

Visual Stimulation

Full-field stimulation was produced with a Ganzfeld stimulator (BigShot Ganzfeld; LKC Technologies, Inc., Gaithersburg, MD, USA) that was equipped with two different types of light source: xenon photostrobe and light-emitting diodes (LEDs). White light flashes at an intensity of 60 phot cd·s/m² or more were generated with the xenon photostrobe. The maximum intensity of the white flash generated with the xenon photostrobe was 826.7 phot cd·s/m². The stimulus intensity was altered by varying the capacitance and applied voltage of a capacitor using software (Ganzfeld control panel; LKC Technologies, Inc.) installed in a personal computer. White light flashes that were below 60 phot cd·s/m², colored-light flashes, and steady background illumination were generated with the following LEDs; red (λ_max = 627 nm), green (λ_max = 530 nm), and blue (λ_max = 470 nm). White flashes were produced by combining the output from these three LEDs. The duration of all stimuli was less than 5 ms. The maximum intensity of white flash generated with the LEDs in the stimulator was 27.3 phot cd·s/m². The stimulus intensity and the background luminance were altered by varying the LED pulse duration using software (Ganzfeld control panel; LKC Technologies, Inc.) installed in the personal computer. The intensities of flashes generated in this system were measured by a calibrated photometer (IL1700; International Light Technologies, Inc., Peabody, MA, USA) and an optical detector (SED033/Y/R; International Light Technologies, Inc.). 

ERG Recording and Analysis

All ERGs described below were recorded from the corneal surface of the left eye of each animal with a bipolar contact lens electrode (H6515NFC; Mayo Corporation, Aichi, Japan). A ground electrode (TN208-016; Unique Medical Co., Ltd., Tokyo, Japan) was attached to the parietal region of the scalp. Responses were amplified 10,000 times and were filtered with a band pass from 0.5 to 1000 Hz. The amplified signals were stored in the evoked potential test equipment (MEB-9104; Nihon Kohden Corporation, Tokyo, Japan). Three to 10 waveforms for each response were averaged. 

Standard Full-Field ERGs.

Standard full-field ERGs were evaluated according to the guideline¹¹ of the International Society for Clinical Electrophysiology of Vision. Following 40 minutes or more of dark adaptation, the rod-driven response (the rod response) and the rod- and cone-driven response (the combined rod-cone response [standard flash]) were elicited by white light flashes at an intensity of 0.009 and 2.8 phot cd·s/m², respectively. Another rod- and cone-driven response (the combined rod-cone response [bright flash]) and oscillatory potentials were simultaneously elicited by white light flashes at an intensity of 17.7 phot cd·s/m². Subsequently, after 10 minutes of light adaptation with a white background light at 29.0 phot cd/m², the single-flash cone response (W/W) and the 30-Hz flicker response were elicited by white light flashes at an intensity of 2.8 phot cd·s/m² under white background light. 

In the waveform analysis, the amplitude and implicit time of the a-wave were measured from baseline to the a-wave trough and from stimulus onset to the a-wave trough, respectively, for the combined rod-cone response and the single-flash cone response. The amplitude and implicit time of the b-wave were measured from the a-wave trough to the b-wave peak and from stimulus onset to the b-wave peak, respectively, for all the responses. For the combined rod-cone response (bright flash), the b/a wave ratio was calculated as the ratio of the b-wave amplitude to the a-wave amplitude. For the oscillatory potentials, the amplitudes of the first and second positive peaks (i.e., OP1 and OP2, respectively) were measured from baseline to each respective peak. The implicit time of the OP1 and OP2 was also measured from stimulus onset to each respective peak. 

a-Wave Analysis.

Immediately before the photopic ERG section of the standard full-field ERGs recording period, responses for the a-wave analysis were recorded in the same manner as that described in our previous reports.^12,13 Briefly, three steps of stimuli were used to elicit the rod- and cone-driven response in the fully dark-adapted state. Thereafter, the cone-driven response in the fully dark-adapted state was elicited by means of a transient rod-saturation procedure, in which the white test stimulus was delivered 500 ms after the conditioning white flash of 4.0 log scot td-s. To elicit the cone-driven response, three steps of test stimuli, which were identical to those used to elicit the rod- and cone-driven response, were used. Rod-isolated responses were obtained by subtracting the cone-driven responses from the rod- and cone-driven responses. To evaluate rod function, the leading edge of the a-wave of the rod-isolated response (i.e., the rod a-wave) was fitted to a curve by the Hood and Birch modification¹⁴ of the Lamb and Pugh model¹⁵:  

 

 

In Equation 1, I is the flash intensity (log scot td-s); t_d is the time delay (ms); t is the time after the flash onset (ms); S is the sensitivity (s⁻²[td-s]⁻¹); and R_max is the maximum response amplitude (μV). The value of t_d was fixed at 3.1 ms, which is the mean value from age-matched healthy monkeys, and S and R_max were varied for the best fit. To evaluate cone function, the leading edge of the a-wave of the cone-driven response (i.e., the cone a-wave) was fitted to a Michaelis-Menten version¹⁶ of Equation 1 combined with an exponential filter:  

 

 

In Equation 2, I is the flash intensity (log phot td-s); t_d is the time delay (ms); t is the time after the flash onset (ms); S is the sensitivity (s⁻³[td-s]⁻¹); and R_max is the maximum response amplitude (μV). The value of t_d was fixed at 1.8 ms, which is the mean value from age-matched healthy monkeys, and S and R_max were varied for the best fit. 

Single-Flash Cone Response (R/B).

Immediately after the photopic ERG section of the standard full-field ERGs recording period, another photopic ERG was elicited by red light flash at an intensity of 2.9 phot cd s/m² under blue background light at 6.9 phot cd/m². For the waveform analysis, amplitude of the b-wave and the photopic negative response (PhNR) were measured from the a-wave trough to the b-wave peak and from baseline to the PhNR trough, respectively. 

Ophthalmoscopy

Immediately after recording the ERGs at each time point, the fundi of both eyes were observed with a binocular indirect ophthalmoscope (HEINE OMEGA 500; HEINE Optotechnik GmbH & Co. KG, Herrsching, Germany) and photographed with a digital fundus camera (GENESIS-D; Kowa Co., Ltd., Aichi, Japan). 

Pathology

Animals were euthanized by exsanguination from the carotid arteries after intramuscular anesthesia with ketamine hydrochloride (Ketalar Intramuscular 500 mg; Daiichi Sankyo Co., Ltd.) and xylazine hydrochloride (Celactal; Bayer Medical Ltd.). Thereafter animals were necropsied and examined macroscopically. The eyes were collected and fixed with Bouin's fluid. The fixed tissues were trimmed, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E). All slides prepared were examined under a light microscope. Retinal findings were recorded separately from the following four retinal regions based on the eccentricity of the retina: 1) central fovea, a region approximately 1 mm wide including the foveola; 2) posterior pole, a region between the central fovea and the equatorial zone; 3) equatorial zone, a region approximately 5 mm wide including the equator of the eyeball; and 4) peripheral zone, a region other than the three retinal regions mentioned above. 

In addition, immunohistochemistry of representative sections was performed using an immunoglobulin conjugated to a peroxidase-labeled dextran polymer (EnVision HRP; Dako Japan, Tokyo, Japan) with 3,3′-diaminobenzidine H₂O₂ as the chromogen. Monoclonal mouse anti-rhodopsin (MAB5316; EMD Millipore, Tamecula, CA, USA) was used as a primary antibody. The sections were then counterstained with toluidine blue. 

Statistics

The predosing values of the ERG parameters for each animal were calculated as the average of the values obtained 15 and 7 days before dosing. For statistical analysis of the ERG parameters in the group given MNU, the paired t-test was used to assess the difference between the values before and 1 day after dosing. The differences were considered to be significant when P was less than 0.05. 

Results

Clinical Observation

In four of six animals treated with MNU, emesis was observed within 1 hour after the completion of intravenous dosing (Table 1). Thereafter, no abnormal clinical signs including any suggestive of visual disturbance were observed in any animal. 

Electroretinogram

Standard Full-Field ERGs.

Typical waveforms of the standard full-field ERGs in vehicle- and MNU-treated monkeys are shown in Figure 1. The amplitude and implicit time of the ERG components before and 1 day after dosing in MNU-treated monkeys are summarized in Table 2. The time course of these ERG parameters after dosing in each animal is shown in Figure 2. 

Figure 1

 

Typical waveforms of the standard full-field ERGs in vehicle- and MNU-treated monkeys. Vehicle or MNU at 40 mg/kg was administered, and the standard full-field ERGs were serially recorded after dosing as described in the text. Arrowheads indicate onset of the light flashes. The responses at baseline (gray traces) are superimposed on those obtained after dosing (black traces). Each trace represents an average of 3 to 10 responses for individual animal.

Table 2

 

Effects of MNU on the ERG Parameters in Monkeys

 

Figure 2

 

Time course of the parameters of the standard full-field ERGs in vehicle- and MNU-treated monkeys. The standard full-field ERGs were serially recorded and analyzed as described in the text. Open symbols with a dashed line and closed symbols with a solid line signify the data from vehicle- and MNU-treated animals, respectively. The number of evaluated MNU-treated animals 1, 3, and 7 days after dosing were six, four, and two, respectively.

MNU gradually attenuated or delayed all of the ERG responses after dosing. In comparison with the predosing values, the following statistically significant differences were detected in the MNU-treated group on the day after dosing: reduced b-wave in the rod response; reduced and delayed a- and b-waves in the combined rod-cone response and the single-flash cone response; decrease in the b/a wave ratio in the combined rod-cone response; reduced and delayed OP1 and OP2 in the oscillatory potentials; and reduced and delayed 30-Hz flicker response. The MNU further aggravated these attenuated responses until 7 days after dosing. 

A-Wave Analysis.

Typical waveforms of the rod a-wave and the cone a-wave in vehicle- and MNU-treated monkeys are shown in Figures 3A and 4A, respectively. The mean values of the a-wave analysis parameters before and 1 day after dosing in MNU-treated monkeys are summarized in Table 2. The time course of the rod and the cone a-wave parameters after dosing in each animal is shown in Figures 3B and 4B, respectively. 

Figure 3

 

(A) Typical waveforms of the rod a-wave in response to various stimulus intensities in vehicle- and MNU-treated monkeys. Vehicle or MNU at 40 mg/kg was administered, and the rod a-waves were derived by subtracting the cone responses from the combined rod-cone responses as described in the text. The dotted lines signify the curves fit from Equation 1. The responses obtained at baseline (gray traces) are superimposed on those obtained after dosing (black traces). (B) Time course of the model parameters of the rod a-wave in vehicle- and MNU-treated monkeys. The model parameters (S, sensitivity; R_max, maximum response amplitude) were determined as described in the text. Open symbols with a dashed line and closed symbols with a solid line signify the data from vehicle- and MNU-treated animals, respectively. The number of evaluated MNU-treated animals 1, 3, and 7 days after dosing were six, four, and two, respectively. Ranges indicated by gray areas signify the 95% CIs based on the values from age-matched healthy cynomolgus monkeys (N = 92).

Figure 4

 

(A) Typical waveforms of the cone a-wave in response to various stimulus intensities in vehicle- and MNU-treated monkeys. Vehicle or MNU at 40 mg/kg was administered, and the dark-adapted cone responses were elicited as described in the text. The dotted lines signify the curves fit from Equation 2. The responses at baseline (gray traces) are superimposed on those obtained after dosing (black traces). Each trace represents an average of six responses for individual animal. (B) Time course of the model parameters of the cone a-wave in vehicle- and MNU-treated monkeys. The model parameters (S, sensitivity; R_max, maximum response amplitude) were determined as described in the text. Open symbols with a dashed line and closed symbols with a solid line signify the data from vehicle- and MNU-treated animals, respectively. The number of evaluated MNU-treated animals 1, 3, and 7 days after dosing were six, four, and two, respectively. Ranges indicated by gray areas signify the 95% CIs based on the values from age-matched healthy cynomolgus monkeys (N = 92). Note that the log R_max was transiently increased in animals given MNU on the day after dosing (asterisk).

In the rod a-wave, MNU decreased both the R_max and S on the day after dosing. The decrease in rod R_max was greater than that in rod S; the mean values of R_max and S on the day after dosing in the MNU-treated group were lower than those obtained before dosing by 0.44 and 0.21 log units, respectively (64% and 38% reduction, respectively). The reduced rod R_max decreased further until 7 days after dosing, whereas the rod S showed no further obvious reduction subsequent to the first day after dosing. Also in the cone a-wave, MNU reduced the S on the day after dosing. On the other hand, MNU transiently increased the cone R_max on the day after dosing and gradually decreased it thereafter until 7 days after dosing. 

Single-Flash Cone Response (R/B).

Typical waveforms of the single-flash cone response (R/B) in vehicle- and MNU-treated monkeys are shown in Figure 5A. The mean values of the ERG parameters for this response before and 1 day after dosing in MNU-treated monkeys are summarized in Table 2. The time course of these ERG parameters after dosing in each animal is shown in Figure 5B. 

Figure 5

 

(A) Typical waveforms of the single-flash cone response (R/B) in vehicle- and MNU-treated monkeys. Vehicle or MNU at 40 mg/kg was administered, and the single-flash cone responses (R/B), elicited with red light flashes under blue background light, were serially recorded. Arrowheads indicate onset of the light flashes. The responses at baseline (gray traces) are superimposed on those obtained after dosing (black traces). Each trace represents an average of 10 responses for individual animal. Note that the PhNR was transiently enhanced on the day after dosing (asterisk) in animals given MNU. (B) Time course of the parameters of the single-flash cone response (R/B) in vehicle- and MNU-treated monkeys. Open symbols with a dashed line and closed symbols with a solid line signify the data from vehicle- and MNU-treated animals, respectively. The number of evaluated MNU-treated animals 1, 3, and 7 days after dosing were six, four, and two, respectively.

On the day after dosing, MNU attenuated the b-wave but transiently enhanced the PhNR. The amplitudes of the b-wave and PhNR gradually decreased thereafter until 7 days after dosing. The PhNR amplitude at 7 days after MNU dosing declined to a value lower than the predosing value. 

Ophthalmoscopy

Typical fundus photographs in a monkey treated with MNU are shown in Figure 6. Funduscopic findings are shown in Table 3. 

Figure 6

 

Typical photographs of the right posterior fundus from a MNU-treated monkey. The MNU at a dose level of 40 mg/kg was administered, and the fundi of both eyes were observed 1, 3, and 7 days after dosing. Note the radial whitish lines surrounding the macula (yellow asterisk) and the increased fundus reflex on the macular area (pink asterisk).

Table 3

 

Funduscopic Findings in MNU-Treated Monkeys

 

Radial whitish lines surrounding the macula were observed in both eyes of all MNU-treated animals on the day after dosing. In addition, both animals that underwent the examination 7 days after dosing showed increased fundus reflex on the macular area in both eyes at this evaluation point. 

Pathology

Typical photomicrographs of the retina in vehicle- and MNU-treated monkeys are shown in Figure 7. Histopathologic findings of the retina are shown in Table 4. 

Figure 7

 

Serial photomicrographs of the retina in a vehicle-treated monkey (A, D, G, J) and MNU-treated monkeys ([B, E, H, K] on the day after dosing and [C, F, I, L] on 7 days after dosing). Photomicrographs (A–C, D–F, G–I, J–L) show the equatorial zone, the posterior pole (excluding the central fovea), the central fovea, and lower magnification of the retina, respectively. On the day after MNU dosing, pyknosis/karyorrhexis (arrowheads) and depletion of the photoreceptor nuclei and shortening and disorientation of the photoreceptor segments (arrows) were the most prominent in the equatorial zone within the retina. In the retina 7 days after MNU dosing, migrating pigment epithelial cells were also observed in the photoreceptor layer (arrow). Note that no abnormal photoreceptors were observed in the central fovea. Stain, H&E, ×400 for photomicrographs (A–I).

Table 4

 

Histopathological Findings of the Retina in MNU-Treated Monkeys

 

Macroscopically, no abnormalities were observed in any animal treated with MNU. Microscopically, animals given MNU showed pyknosis and karyorrhexis of the photoreceptor nuclei in all the retinal regions except for the central fovea on the day after dosing (Figs. 7B, 7E, 7H); in the posterior pole excluding the central fovea, the abnormal photoreceptor nuclei were localized in the inner region of the outer nuclear layer. These findings were more severe in the equatorial zone and the posterior pole compared with those in the peripheral zone. Furthermore, depletion of the photoreceptor nuclei and shortening and disorientation of the photoreceptor segments were noted only in the equatorial zone, suggesting that the equatorial retina was primarily involved. In the retinas from animals necropsied 3 days after MNU dosing, the lesions that had been observed only in the equatorial retina on the day after dosing became severe and spread to the posterior pole; depletion of the photoreceptor nuclei and shortening and disorientation of the photoreceptor segments were found, in addition to the findings seen on the day after dosing. In these two animals, migration of pigment epithelial cells was observed in the photoreceptor layer (data not shown). In the retinas of animals necropsied 7 days after MNU dosing, the lesions mentioned above were basically sustained, and the incidence of the pyknotic and karyorrhectic cells decreased presumably due to the degenerative process. Additionally, cystoid degeneration in the inner nuclear layer of the posterior pole and in the outer plexiform layer of the central fovea, considered to be a secondary reaction to the rest of the retinal lesions, was observed (Figs. 7F, 7I, 7L). No remarkable findings were observed in the other tissues of the eye such as the cornea, iris, ciliary body, lens, or optic nerve in any animal. 

Typical photomicrographs of immunohistochemical findings are shown in Figure 8. In the retina from a vehicle-treated animal, most of the photoreceptor cells showed positive reaction for rhodopsin around the nuclei in the equatorial zone (Fig. 8A). In this area, dense and well-ordered positive reaction was seen in the photoreceptor layer. In the posterior pole excluding the central fovea, the rhodopsin-positive reaction was localized in the inner region of the outer nuclear layer and the photoreceptor layer (Fig. 8D). In contrast, there was no positive reaction in the central fovea (Fig. 8G). In the retinas from MNU-treated animals necropsied on the day after dosing (Figs. 8B, 8E, 8H), the occurrence and localization of the rhodopsin-positive photoreceptors was generally consistent with those of the vehicle-treated animal. In the retinas from animals necropsied 7 days after MNU dosing, the positively stained photoreceptors were decreased especially in the equatorial zone (Fig. 8C), which corresponded to the decreased number of the photoreceptor cells, as observed in the H&E staining. These immunohistochemical findings suggested that majority of the degenerated photoreceptors were the rods. In addition, the intensity of the positive reaction for rhodopsin was enhanced in the outer plexiform layer of the posterior pole excluding the central fovea (Fig. 8F), suggesting a redistribution of rhodopsin within the rod cell body in response to the outer segment damage. 

Figure 8

 

Photomicrographs of immunohistochemistry for rhodopsin in a vehicle-treated monkey (A, D, G) and MNU-treated monkeys ([B, E, H] on the day after dosing and [C, F, I] on 7 days after dosing). Photomicrographs (A–C, D–F, G–I) show the equatorial zone, the posterior pole (excluding the central fovea), and the central fovea, respectively. Note that rhodopsin-positive reaction of the outer nuclear layer was localized in its inner region in the posterior pole (excluding the central fovea). There was no positive reaction in the central fovea. Immunohistochemistry for rhodopsin, ×400.

Discussion

We electrophysiologically and histopathologically characterized MNU-induced retinal changes in monkeys and identified both similarities and differences to those in patients with RP, a set of hereditary retinal diseases that feature degeneration of the photoreceptors.¹⁷ To our knowledge, this report represents the first description of both functional and morphologic alterations of the retina induced by MNU in monkeys. 

Rod System

In the present study, the dark-adapted ERGs were acutely diminished, indicating acute and severe dysfunction of the rod pathway of the retina. For an in-depth investigation into the functional effects of MNU on the rod photoreceptors, we conducted an a-wave analysis based on the method¹⁴ by Hood and Birch. MNU markedly depressed the maximum response parameter (R_max) of the rod a-wave in the monkeys on the day after dosing. This finding strongly suggested a decrease in the number of functioning rod photoreceptors in a rapid manner, consistent with the rod-dominant photoreceptor degeneration in histopathology of the present study. In RP patients, the characteristic ERG change is a progressive and consequently severe attenuation in the rod-driven responses.¹⁷ Therefore, MNU quite acutely induced the rod deficit in monkey retinas, which was similar to that in patients with RP except for the rapid time course. 

A similarity to RP from the viewpoint of relative integrity of phototransduction in the rods was identified in the retinas of MNU-treated monkeys. The MNU decreased the sensitivity parameter (S) of the rod a-wave in addition to the rod R_max in this study; the decrease in the rod S was milder than that in the rod R_max (Fig. 3B). The less affected rod S in comparison with rod R_max has been reported in patients with RP.¹⁸ The S value mainly provides a measure of the efficiency of the phototransduction amplification process.¹⁹ Therefore, the phototransduction process in the rods is probably preserved at some level in MNU-treated monkeys, as well as in patients with RP. It has also been reported that MNU-induced retinal damage in rats was attributed to DNA adduct formation restricted to photoreceptor cell nuclei.² Taken together, the lesser effect on the S parameter in this study may be explained in terms of the mechanism of MNU-induced photoreceptor toxicity (i.e., the outer segment, where phototransduction takes place, is not the primary target). 

In terms of a functional involvement of postreceptoral retinal components, a clear difference to RP patients was identified in MNU-treated monkeys. MNU decreased the b/a wave ratio of the dark-adapted ERG in the present study, suggesting that the rod bipolar cells were more compromised than the rod photoreceptors. This finding was a difference to typical RP, in which the outer retina is mainly disrupted.¹⁷ MNU recently has been reported to cause primary damage in the retinal vasculature, indicated by immunohistochemistry in the mouse retina.⁹ Therefore, the impaired bipolar cell function in this study could be attributed to decreased blood supply in the inner retina related to an effect on retinal vasculature, although this study lacks detailed evaluations of the retinal blood vessels such as by fluorescein fundus angiography. 

Death of the rods in RP patients usually begins in the mid peripheral (equatorial) retina and progresses with time to involve the macula and more peripheral retina.²⁰ Histopathologically, we found similar characteristics like expansion of the rod involvement within the retina after MNU administration to monkeys, although the time course was fairly short compared with that in RP. This similarity between humans with RP and MNU-treated monkeys could be attributable to the similarity in rod and cone cell distribution within the retina between humans and monkeys.²¹ In contrast, it has been reported that photoreceptor cell death started in the posterior pole and spread peripherally in mice²², rats,²³ and hamsters.⁶ Therefore, although the exact mechanisms underlying this species difference are unclear, MNU-induced RP model in monkeys may turn out to be more clinically relevant than that in other species from the standpoint of initiation and expansion of the rod deterioration within the retina. 

Cone System

In patients with RP, degeneration of the photoreceptors primarily occurs in the rods, and therefore, the fovea, where the cones are tightly packed, is generally the last region of the retina to deteriorate.²⁰ We also identified the difference in susceptibility between the rod and cone photoreceptors in monkeys given MNU. Namely, no obvious degeneration of the cone photoreceptors was detected by light microscopy, despite the finding that the rod photoreceptors remarkably degenerated. Meanwhile, MNU diminished the cone-driven ERGs and the rod-driven ERGs. As one of the limitations in this study, we did not perform a multifocal or focal ERG, which could provide the correlation between functional and morphological cone photoreceptor topography. Therefore, the finding of no visible cone degeneration despite a functional impairment in MNU-treated monkeys remains largely unexplained. Also, this finding in the cone photoreceptors is a major difference to human RP. 

An interesting finding from a perspective of functional effect of MNU on the cones was detected in this study by means of the a-wave analysis, which is specialized to the photoreceptors. The MNU transiently increased the cone R_max, which is a model parameter that generally provides an estimation of the number of functioning cones.¹⁹ The exact mechanisms responsible for this phenomenon remain to be elucidated. However, this might be explained by a disinhibition of the cones from acutely degenerating rods, based on a rod-cone interaction hypothesis.²⁴ Another possible explanation is that the increased cone R_max was associated with the transiently augmented electrical activity from the inner retina, indicated by the simultaneously enhanced PhNR as discussed below. 

Inner Retina

In the present study, no marked histopathologic abnormalities were observed in areas of the retina proximal to the photoreceptors in light microscopy. Similar characteristics in MNU-induced retinal lesions have also been reported to occur in a variety of animal species.¹ Therefore, MNU is suitable as a research tool to acutely provoke selective degeneration of the photoreceptors in the retina of experimental animals. 

From the aspect of function, we observed an interesting ERG finding in the present study; MNU transiently enhanced the PhNR, an ERG waveform component that has been shown to originate mainly from the retinal ganglion cells (RGCs) in monkeys.²⁵ This result suggests that electrical activity of the inner retina including the RGCs was transiently augmented or relatively preserved within the retina. 

Several differences to RP patients were identified in the retinas of MNU-treated monkeys in this study. The fundus in human RP typically shows intraretinal pigmentation, which is histopathologically the retinal pigment epithelial cells (RPEs) migrating to perivascular sites in the inner retina.^18,20 We found neither fundi with pigment deposits nor RPEs migrating to the inner retina in histopathology in this study. Alternatively, the fundus oculi of all the MNU-treated monkeys showed radial whitish lines surrounding the macula, although the corresponding histopathologic change was not identified. This fundus appearance is not the characteristic one observed in patients with RP and is rather similar to that in patients with toxic retinopathy induced by chloroquine in terms of a remarkable involvement of the macular area (i.e., bull's-eye maculopathy).²⁶ The exact causes of these differences in fundus oculi between the monkeys and humans with RP are unclear. However, they could be in part because MNU quite acutely induces retinal degeneration, whereas human RP slowly progresses by decades. 

The present study has limitations as described below. First, no long-term follow-up examinations were conducted after the MNU treatment. Second, we set only one dose of MNU (40 mg/kg), which was considered as the maximum tolerable dose in monkeys based on our preliminary study (data not shown). This made it difficult to produce more severe photoreceptor degeneration in monkeys with nondetectable ERGs. These limitations are critical if the MNU-induced retinal lesion in monkeys is utilized to test new treatment strategies such as cell transplantation and retinal prosthesis. Therefore, it would be ideal if a refined method were developed where the severe photoreceptor degeneration is induced only in one eye by an intravitreal injection of MNU in monkeys, just as reported to occur in mice.⁸ This approach would also solve a possible ethical problem to conduct long-term experiments in monkeys with severely impaired vision. An investigation of the long-term effect of MNU on the monkey retina is desired, because the present study revealed several aforementioned aspects of MNU-induced acute photoreceptor degeneration in monkeys, which might be inappropriate as an animal model of RP. 

Conclusions

Our results indicated that MNU acutely induced rod-dominant photoreceptor degeneration in monkey retinas, but the photoreceptor function was impaired in the cones and in the rods. Functional involvement of the postreceptoral components was also indicated. 

Acknowledgments

The authors thank Hidetaka Kudo and Eiichiro Nagasaka of Mayo Corporation, and Yuka Ofune of Daiichi Sankyo RD Novare Co., Ltd., for technical assistance. 

Disclosure: J. Kinoshita, None; N. Iwata, None; T. Maejima, None; M. Imaoka, None; T. Kimotsuki, None; M. Yasuda, None