内 容 要 旨 目 次
主 論 文
Photoelectric dye used for Okayama University-type Retinal Prosthesis (OURePTM)
reduces the apoptosis of photoreceptor cells(光電変換色素の神経アポトーシス抑制効果)

劉 詩卉, 松尾俊彦, 細谷 修, 内田拓也

Journal of Ocular Pharmacology and Therapeutics （掲載予定）
平成 27 年 5 月 2016 The Annual Meeting of the Association for Researchin Vision and Ophthalmology(ARVO)学会総会に発表

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Photoelectric dye used for Okayama University-type
Retinal Prosthesis (OURePTM) reduces the apoptosis ofphotoreceptor cells(光電変換色素の神経アポトーシス抑制効果)[緒言] Introduction
Retinitis pigmentosa is a hereditary disease that involves the loss of retinal photoreceptor
cells which perceive light. Patients with retinitis pigmentosa experience slowly progressive
constriction of the visual field, caused by peripheral retinal dystrophy, and finally lose the visionby macular involvement. The speed of deterioration differs largely from patient to patient2.
In blind patients with retinitis pigmentosa who have lost all photoreceptor cells, treatment
strategy is to replace dead photoreceptor cells with retinal prosthesis. Several types of retinal
prosthesis have been developed recently to replace the lost photoreceptor cells. The prevailingtypes of retinal prosthesis output electric current in correspondence to camera-captured andprocessed image, and stimulate the remaining retinal neurons which send axons to the brain. Other
types of retinal prosthesis utilize photovoltaic systems7 or wireless subretinal neurostimulator8.
Our system used photoelectric dye molecules to replace dead photoreceptor cells.
A photoelectric dye is an organic molecule which generates electric potential in response tolight. Photoelectric dye-coupled polyethylene thin films (dye-coupled films), called Okayama
University-type retinal prosthesis (OURePTM), induced neuronal response at light stimulation. Our
previous studies showed that OURePTM could reduce neuronal apoptosis in the eyes of Royal
College of Surgeons (RCS) rats. In this study, we tested whether intravitreous injection of thephotoelectric dye would rescue photoreceptor cells from the apoptosis in RCS rats.
[材料と方法]Materials and Methods

Animals and experimental design
Photoelectric dye molecules, 2-[2-[4-(dibutylamino) phenyl] ethenyl]-3carboxymethylbenzothiazolium bromide (503.5 of molecular weight, NK-5962, Hayashibara, Inc.,
Okayama, Japan, Fig. 1A), were dissolved in distilled deionized water (Fig. 1B) at theconcentration of 8.2 μg/ml (16 μM). This original stock solution (8.2 μg/ml) was diluted withsaline (0.9% sodium chloride) to make a series of ten-fold dilutions from 8.2×10-4 μg/ml to 8.2
μg/ml. Twenty male pink-eyed RCS rats (RCS/Jcl-rdy/rdy, p-, CLEA Japan, Inc., Tokyo) at theage of 4 weeks were assigned to five groups (n=4) which received the dye at 5 different
concentrations. Intravitreous injection was done twice at the age of 4 weeks and 5 weeks (Fig.
1D). This study was approved by the Animal Care and Use Committee at Okayama University,
based on the Animal Welfare and Management Act in Japan. All experimental procedures were inaccordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
RCS rats were anesthetized by intraperitoneal injection of ketamine (87 mg/kg body weight)
and xylasine (13 mg/kg). Mydriasis was induced by 0.5% tropicamide and 0.5% phenylephrine
instillation, and corneal anesthesia was further obtained with 0.4% oxybuprokaine instillation.
Under a dissecting microscope, 3 μl of dye solution and saline were injected into the vitreous of
the left eye and the right eye, respectively, with a 30-gauge needle attached to a Hamilton syringe(50 μL 1705 LT SYR, Hamilton Company, Reno, NV, USA). The needle was inserted almost at aperpendicular angle, 1 mm from the corneoscleral limbus to avoid the damage to the lens. Next,
antibiotic (0.5% moxifloxacin) eye drops were instilled to both eyes. Each rat was housed in astandard rat cage in the 12-hour-each light and dark cycle at the Animal Center of Okayama
University. In a separate experiment, three rats were placed and maintained in constant darkness
after intravitreous injection of the dye at the concentration of 8.2 μg/ml (16 μM).
Immunohistochemistry2

At the age of 6 weeks, 2 weeks after the first injection, rats were sacrificed by an overdoseof ether. The eyes were enucleated and fixed in 4% paraformaldehyde for 2 to 3 hours, cut into
halves circumferentially in the midperiphery of the eye balls, and cryo-protected by immersion in
10% sucrose in 100 mM phosphate buffer (pH 7.4) for 3 hours. Serial frozen sections with 14 μm
thickness were cut parallel to the vertical meridian of the eye at the optic nerve head, andimmunohistochemical staining was done as described previously. The primary antibodies wereanti-glial fibrillary acidic protein (GFAP) in 1:200 dilution (mouse monoclonal antibody,
Chemicon, Temecula, CA, USA), and anti-protein kinase Cα (PKCα) in 1:250 dilution (mousemonoclonal antibody, Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 594 goat anti-mouse IgG
was used as a fluorescence-conjugated secondary antibody. Nuclei were counterstained with 4′,6diamidino-2-phenylindole (DAPI). The tissue was covered with anti-fade reagents (ProLong
Antifade Kit, Invitrogen, Waltham, MA, USA) and air-dried.
All images were captured at the constant exposure time with a frame (438 μm x 330 μm) byan Olympus FSX100 microscope, using 20x NA0.75 objective lens (Olympus, Tokyo, Japan), andsaved as 24-bit RGB TIFF files. Alexa Fluor 594 and DAPI signals were sequentially detectedwith Olympus filter sets U-MWIG3 and U-MNUA2. GFAP or PKCα immunoreactivity wasassessed in four retinal sites: “a” and “d”, beginning at 373 μm superiorly and inferiorly from theoptic nerve head, respectively, and sites “b” and “c”, beginning at 160 μm superiorly andinferiorly from the optic nerve head, respectively (Fig. 1C). Five photographs in each of fiveretinal vertical sections per eye were taken, and an entire frame of each photograph with 20x
objective lens was used for the following measurements.
RGB images were imported into the Image J software (National Institutes of Health,Bethesda, MD, USA), and converted to 8-bit grayscale images. For background subtraction, themean background staining was defined by measuring an area positioned away from specificimmunoreactive signals. After the background subtraction, the total area of immunoreactivity wasdetermined by using a standardized histogram-based technique. To define a pixel as
immunoreactive, constant threshold values were set to 50 for GFAP and 55 for PKCα
immunoreactivity, respectively, and the number of pixels that matched or exceed each set
threshold value was calculated for each image. Finally, the percentage of immunoreactive area
was calculated from total immunoreactive area in a given size frame (height 193 μm, width 173μm) at four predefined sites per retinal section (sites a, b, c, and d), and the data were compared by
two-factor analysis of variance (ANOVA).
Apoptosis detection and retinal thickness measurement
Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediatedfluorescein-conjugated-dUTP nick-end-labeling (TUNEL) assay, according to the manufacturer’s
instructions (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). The
images at the constant exposure time were captured by an Olympus FSX100 microscope,equipped with the fluorescent filter cube U-MWIBA3. The areas for TUNEL assay were the sameas those for immunohistochemical staining (sites a, b, c, and d). Five photographs in each of fiveretinal vertical sections per eye were taken, and an entire frame (220 μm x 165 μm) of eachphotograph with 40x NA0.95 objective lens was used for measurements. The number of TUNELpositive cells in the inner nuclear layer and the outer nuclear layer was counted in each frame ofphotographs, and the thickness of the inner and outer nuclear layer was measured. The number of
TUNEL-positive cells per 1,000 μm2 of the inner nuclear layer or the outer nuclear layer was
calculated for comparison by two-factor ANOVA.

[結果] Results

TUNEL staining and retinal thickness

TUNEL-positive signals were compared among the left eyes with dye injection at different
concentrations and the right eyes with control saline injection. Under 12-hour light/dark cycle
condition, in saline-injected eyes, numerous TUNEL-positive signals were detectable in the outer
nuclear layer where the nuclei of photoreceptor cells were located. In contrast, TUNEL-positive
cells appeared to decrease in all 4 different sites (a, b, c, and d) of retinal sections from dyeinjected eyes (Fig. 2). TUNEL-positive cells in the outer nuclear layer were significantly different
among different concentrations of the dye (P=0.0001), but not significantly different among 4
different retinal sites (P=0.144, two-factor ANOVA, Fig. 3). TUNEL-positive cells in the outer3

nuclear layer of dye-injected eyes were significantly less at 3 higher concentrations of the dye,
ranging from 16 μM to 16 x 10-2 μM, compared with saline-injected eyes (P=0.0001, P=0.0001,
and P=0.002, post-hoc test, the least significant difference, Fig. 3).
In contrast, the number of TUNEL-positive cells in the inner nuclear layer was neither
significantly different among different concentrations of the dye (P=0.084) nor among 4 different
retinal sites (P=0.927, two-factor ANOVA, Fig. 4). However, post-hoc test showed significant
decrease of the number of TUNEL-positive cells in the inner nuclear layer only at the highest
concentration of the dye (16 μM), compared with saline (P=0.043, Fig. 4).
Morphometric analysis showed that the thickness of the outer nuclear layer was
significantly different among different concentrations of the dye (P=0.0001) and among 4
different retinal sites (P=0.014, two-factor ANOVA, Fig. 5). The outer nuclear layer was
significantly thicker in the dye-injected eyes at higher concentrations, ranging from 16 μM to 16 x
10-2 μM, compared with saline-injected eyes (P=0.001, P=0.001, and P=0.026, post-hoc test, Fig.
5), under 12-hour light/dark cycle condition. In addition, significant difference was noted in the
thickness of the inner nuclear layer among different concentrations of the dye (P=0.014) andamong 4 different retinal sites (P=0.0001, two-factor ANOVA, Fig. 6), under 12-hour light/darkcycle condition. However, post-hoc tests did not reach significance.
In another series of experiments, RCS rats injected with saline and dye at a highest
concentration (16 μM) were kept under constant dark condition. The number of TUNEL-positive
cells in the outer nuclear layer was significantly different between dye injection and saline
injection (P=0.0001), but not significantly different among 4 different retinal sites (P=0.982, twofactor ANOVA). As post-hoc tests, the number of TUNEL-positive cells in the outer nuclear layerof the dye-injected eyes was significantly less than in the saline-injected eyes (P=0.0001), but nosignificant difference was noted in the number of TUNEL-positive cells between the constant dark
condition and the 12-hour light/dark cycle (P=0.411, Fig. 2 and 3). The number of TUNELpositive cells in the inner nuclear layer showed no significant difference between the dye-injectedeyes and saline-injected eyes (Fig. 4).
Morphometric analysis showed that the outer nuclear layer tended to be thicker in the dyeinjected eyes compared with saline-injected eyes under constant dark condition, although thedifference was not significant (P=0.054, Fig. 5). No significant difference was noted in the
thickness of the inner nuclear layer between the dye-injected and saline-injected eyes, underconstant dark cycle condition (P= 0.145, Fig. 6).

GFAP immunohistochemistry

GFAP-immunoreactive signals were compared among the left eyes with dye injection at
different concentrations and the right eyes with control saline injection. In the saline-injected eyes,
strong GFAP-immunoreactivity was present at Müller cells in all sites of the retina and was
mainly localized on their end-feet which spread to the ganglion cell layer. In addition, radial
Müller cell processes, running in the inner plexiform layer, showed intense GFAPimmunofluorescence (Fig. 7). In the dye-injected eyes, immunohistochemical localization of
GFAP in the retina was basically similar to that observed in the saline-injected eyes (Fig. 7). At acloser look, many Müller cell radial processes, exhibiting weak and discontinuous staining, werenoted in the inner plexiform layer (Fig. 7).
Semi-quantitative analysis showed that GFAP-immunoreactive areas were significantly
different among different concentrations of the dye (P=0.0001) and also among 4 different retinal
sites (P=0.0001, two-factor ANOVA, Fig. 8). GFAP-immunoreactive areas significantly decreased
at two concentrations of the dye (16 μM and 16 x 10-2 μM) in the dye-injected eyes, comparedwith the saline-injected eyes (P=0.023 and P=0.007, post-hoc test, Fig. 8).

PKCα immunohistochemistry

In dye-injected eyes, PKCα immunoreactivity was evident in dendritic terminals of rod bipolar cells
in the outer plexiform layer, somata in the inner nuclear layer, and axons, running perpendicularly
throughout the inner plexiform layer, as well as the axon terminals located at the border between
the inner plexiform layer and ganglion cell layer. The patterns of PKCα staining in the retina of thedye-injected eyes appeared to be different from those in the saline-injected eyes (Fig. 7). PKCαimmunoreactive areas were significantly different among different concentrations of the dye(P=0.01), but not significantly different among 4 different retinal sites (P=0.413, two-factor
ANOVA, Fig. 9). Post-hoc tests showed significant increase of PKCα-immunoreactive areas at 3
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higher dye concentrations, ranging from 16 μM to 16 x 10-2 μM, compared with saline injection(P=0.002, P=0.003, and P=0.014, respectively).
[考察] Discussion
The present study aimed to investigate whether the photoelectric dye has protective effect on
retinal neurons. We used RCS rats as an animal model which show progressive photoreceptor
degeneration as the consequence of a primary RPE gene mutation. Our results demonstrated that
repeated intravitreous injection of the photoelectric dye provided the protection of the outer
nuclear layer neurons at the earlier stage of dystrophy in RCS rats both under the 12-hour
light/dark cycle and under the 24-hour continuous dark condition.
Many potential agents have been tested in animal models for their ability to protect
photoreceptor cells from death. Ciliary neurotrophic factor (CNTF) has entered into a phase I
clinical trial in human patients with retinitis pigmentosa24. Our previous studies showed that thephotoelectric dye-coupled films (OURePTM) could reduce neuronal apoptosis. In addition, thephotoelectric dye could prevent retinal cells from death in mixed culture of glial cells and retinal
neurons in vitro. We, therefore, speculated that the photoelectric dye might become a newneuroprotective agent.
Because the lens is relatively large in the small space of the eyeball of the rats, repeatedintraocular injections would cause cataract formation and vitreous hemorrhage. We took care to
reduce the incidence of cataract formation and vitreous hemorrhage after intravitreous injection. In
the present study, the photoelectric dye was administered intravitreally twice at 4 weeks and 5
weeks of the age in RCS rats, and the rats were observed in the following 7 days. In the strain of
RCS rats, used in this study, photoreceptor cells begin to degenerate on the 22th day after thebirth, and the degeneration proceeds rapidly toward the 32th day. In a previous study, TUNELlabeled photoreceptor cells suddenly appeared on 25 to 30 days, and the majority of thephotoreceptor nuclei was TUNEL-labeled on 35 days of age. Few photoreceptor nuclei remain in
the outer nuclear layer by the 60th day. We, therefore, chose the age of 4th week for the timing of
injection to determine whether the photoelectric dye was effective to prevent photoreceptor cells
from apoptotic death.
The treatment with the dye under 24-hour continuous dark condition also significantly reduced
apoptotic cells. Furthermore, we found no difference in the number of retinal apoptotic cells
between the 24-hour continuous dark condition and the 12-hour light/dark cycle. However, aprevious study reported that dark condition could delay the deterioration of the dystrophic rat
retina and that the animals in a dark environment showed less deterioration of theelectroretinographic response. Further studies would be required to explain the contradictory
results between the previous study and the present study, concerning the reduction of retinal
apoptosis under constant dark and light/dark cycle condition.
GFAP is a constituent of glial filaments in astrocytes. In the central nervous system, astrocytes
respond in an active manner when they suffer from diseases, trauma, chemical insults, and genetic
disorders. Enhanced expression of GFAP is considered a marker of gliosis and hypertrophy of
glial cells. Therefore, we examined GFAP immunoreactivity in retinal Müller cells after
intravitreous dye injection. We found that intravitreous injection of the dye led to direct or indirect
downregulation of GFAP expression, and might inhibit glial injury reaction. The variability in thesemi-quantitative image analysis resulted in significant differences in GFAP-staining for someconcentrations of the dye, but not for others. Nevertheless, the results suggest that photoelectric
dye may downregulate GFAP expression and inhibit glial cell activation.
Rod bipolar cells are second-order retinal neurons that make synapses with rod photoreceptors,
and their axon terminals are located in the inner plexiform layer, close to the ganglion cell layer.
Because intraocular administration of the photoelectric dye showed inhibitory effect on
photoreceptor cell apoptosis, we wondered whether intravitreous dye injection would also preventrod bipolar cell dysfunction in RCS rats. Thus we performed immunohistochemical staining of
PKCα, a rod bipolar cell marker. Dendrites of rod bipolar cells are the major phosphorylation sitesof PKCα, and PKCα upregulation would indicate the improvement of bipolar cell function. Ourdata revealed that the atrophy of dendrites was correlated with decreased PKCα immunoreactivity
in both dye-injected eyes and saline-injected control eyes. Furthermore, we observed significant
increase of PKCα immunoreactivity in the eyes injected with higher concentrations of the dye.
These data suggest that the photoelectric dye may help prevent rod bipolar cell dysfunction in
RCS rats.
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Previous studies by different groups showed that subretinal implantation of materials could
slow down retinal degeneration. We initially considered that photoelectric dye-coupled
polyethylene films in subretinal space of RCS rats’ eyes would exert a similar protective effect on
the retina, as described for other materials. The present study revealed that the photoelectric dye
could slow down the process of retinal degeneration in RCS rats. The therapeutic effect ofphotoelectric dye on retinal protection, either dependent or independent of light, remains to be
determined. Behavioral tests might help evaluate the efficacy of the dye in RCS rats, as performed
previously in RCS rats with the dye-coupled film implantation.
A major limitation in this study is that we examined the apoptosis by TUNEL assay and thethinning of the outer nuclear layer in the ongoing process of retinal dystrophy at a rather earlier
stage in RCS rats. Further studies need focus on the late stage of retinal dystrophy to test longterm efficacy of the photoelectric dye on overall degeneration of the retinal layers. In addition, it
remains unclear how the photoelectric dye prevents photoreceptor degeneration. Further studies
are required to elucidate the underlying molecular mechanism of the apoptosis reduction. Semiquantitative measurements of GFAP and PKCα-immunoreactivity, as a limitation in the method,
would explain inconsistent results in statistical analyses of measurements.
[結論] Conclusion
our results show that the injection of the photoelectric dye could effectively reduce the apoptosisof photoreceptor cells and downregulate GFAP expression. In the present study, we showed thethreshold of the concentration for preventing the photoreceptor degeneration. The best concentration
would be obtained from a cell culture experiment at the next step. We confirmed biological safetyof the photoelectric dye in all tests, including cell safety test, Ames test, chromosomal aberration
test, eye irritation test, skin sensitivity test, acute (a single injection) and subacute (28-day repeatedinjection) systemic toxicity test (data not shown). The photoelectric dye might be a candidate agent
for slowing photoreceptor degeneration. In addition, retinal prosthesis, OURePTM, would bepositioned as a medical device with pharmacological effect to prevent neuronal apoptosis in theretina.

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