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In this study, we aimed to determine the electrophysiological efficacy, safety, and electrical stability of a chronically implanted electrode for suprachoroidal transretinal stimulation (STS) in rabbit eyes. A platinum microelectrode was implanted into the scleral pocket of rabbit eyes (n = 5) and followed-up for 6 months. To evaluate the electrophysiological efficacy, electrically evoked potentials (EEPs) were measured every month after implantation. To evaluate safety, fundus examinations, fluorescein angiograms, electroretinograms (ERGs), and visually evoked potentials (VEPs) were measured before and every month after the implantation. At the end of the experiment, histological examination of retinal tissue beneath the site of the electrode was performed. To evaluate electrical stability, the resistance of the circuit was measured every month after implantation. EEPs could be elicited from the STS electrodes at all testing times. The mean threshold current to evoke EEPs was 186.4 ± 47.0 µA at 6 months after implantation. There was no significant change in the threshold over the follow-up period. The resistance of the circuit was significantly increased at 1 months after implantation, with no further increase at 6 months. There was no statistically significant change in the relative amplitudes and implicit times of a- and b-waves of ERGs and VEPs. No intraocular infection, inflammation, or vitreoretinal proliferation was observed in any eye. Histological examination revealed no retinal damage beneath the electrode. We conclude that chronically implanted electrodes for STS appear to be effective, safe, and electrically stable.

Purpose: To evaluate the improvement in targeted reaching movements toward targets of various contrasts in a patient implanted with a suprachoroidal-transretinal stimulation (STS) retinal prosthesis., Methods: An STS retinal prosthesis was implanted in the right eye of a 42-year-old man with advanced Stargardt disease (visual acuity: right eye, light perception; left eye, hand motion). In localization tests during the 1-year follow-up period, the patient attempted to touch the center of a white square target (visual angle, 10°; contrast, 96, 85, or 74%) displayed at a random position on a monitor. The distance between the touched point and the center of the target (the absolute deviation) was averaged over 20 trials with the STS system on or off., Results: With the left eye occluded, the absolute deviation was not consistently lower with the system on than off for high-contrast (96%) targets, but was consistently lower with the system on for low-contrast (74%) targets. With both eyes open, the absolute deviation was consistently lower with the system on than off for 85%-contrast targets. With the system on and 96%-contrast targets, we detected a shorter response time while covering the right eye, which was being implanted with the STS, compared to covering the left eye (2.41 ± 2.52 vs 8.45 ± 3.78 s, p < 0.01)., Conclusions: Performance of a reaching movement improved in a patient with an STS retinal prosthesis implanted in an eye with residual natural vision. Patients with a retinal prosthesis may be able to improve their visual performance by using both artificial vision and their residual natural vision., Clinical Trial Registration: Beginning date of the trial: Feb. 20, 2014 Date of registration: Jan. 4, 2014 Trial registration number: UMIN000012754 Registration site: UMIN Clinical Trials Registry (UMIN-CTR) http://www.umin.ac.jp/ctr/index.htm.

Purpose: To determine the feasibility of a 49-channel suprachoroidal-transretinal stimulation (STS) retinal prosthesis that was implanted for 1 year., Methods: Three patients with advanced retinitis pigmentosa (RP) and with less than or equal to hand motion (HM) vision were studied. A 49-channel STS system was implanted in a scleral pocket, and the stability of the prosthesis, adverse events, and its efficacy were investigated., Results: The electrode array was implanted in a scleral pocket located under the parafoveal area through a scleral incision at 14 mm in patient (Pt) 1, 16 mm in Pt 2, and 18 mm in Pt 3 from the sclerocorneal limbus. No complications occurred during surgery in all cases. During the follow-up period, iridocyclitis developed in Pt 2 and Pt 3, which was successfully treated with topical medications. The implanted devices continued functioning and elicited phosphenes in all cases. The results of localization tests (P < 0.05) and table tests (P < 0.05) were significantly better with the prosthesis turned on than turned off in Pt 3. The deviations of the walking tests were smaller with the prosthesis turned on than off in Pt 2 and Pt 3 at multiple times after the implantation., Conclusions: The 49-channel STS retinal prosthesis was able to elicit phosphenes in all patients with advanced RP for the entire 1-year experimental period without major complications. Better results on visual tasks were found in the eyes in which the electrode array was implanted closer to the fovea centralis.

To investigate the changes in the wavefront aberrations and pupillary shape in response to electrical stimulation of the branches of the ciliary nerves in cats. Seven eyes of seven cats were studied under general anesthesia. Trains of monophasic pulses (current, 0.1 to 1.0 mA; duration, 0.5 ms/phase; frequency, 5 to 40 Hz) were applied to the lateral or medial branch of the short ciliary nerve near the posterior pole of the eye. A pair of electrodes was hooked onto one or both branch of the short ciliary nerve. The electrodes were placed about 5 mm from the scleral surface. The wavefront aberrations were recorded continuously for 2 seconds before, 8 seconds during, and for 20 seconds after the electrical stimulation. The pupillary images were simultaneously recorded during the stimulation period. Both the wavefront aberrations and the pupillary images were obtained 10 times/sec with a custom-built wavefront aberrometer. The maximum accommodative amplitude was 1.19 diopters (D) produced by electrical stimulation of the short ciliary nerves. The latency of the accommodative changes was very short, and the accommodative level gradually increased up to 4 seconds and reached a plateau. When only one branch of the ciliary nerve was stimulated, the pupil dilated asymmetrically, and the oblique astigmatism and one of the asymmetrical wavefront terms was also altered. Our results showed that the wavefront aberrations and pupillary dilations can be measured simultaneously and serially with a compact wavefront aberrometer. The asymmetric pupil dilation and asymmetric changes of the wavefront aberrations suggest that each branch of the ciliary nerve innervates specific segments of the ciliary muscle and dilator muscle of the pupil.

Purpose: Our goal was to determine the feasibility of using electrochemically treated bulk platinum electrodes with large charge injection capacity for a retinal prosthesis., Methods: Seven eyes of seven cats were studied. Small retinal areas were focally stimulated with electrochemically treated bulk electrodes (φ = 500 µm) placed in a scleral pocket. Fundus images with near-infrared (800-880 nm) light were recorded, and a 2D map of the reflectance changes elicited by the electrical currents was constructed by subtracting the images taken before stimulation from those taken after stimulation. The impedance of each electrode was measured at 1 kHz. The degree of retinal elevation by the electrode was measured by optical coherence tomography. Scleral thickness where the electrode array was inserted was measured in histologic sections., Results: The diameter of reflectance changes (full width at half maximum) was 0.42 ± 0.22 mm [mean ± standard deviation (SD)] in minor axes and 1.46 ± 0.82 mm in major axes. The threshold current decreased with a reduction in the residual scleral thickness (R (2) = 0.9215; P = 0.0002); it also decreased with an increase in retinal elevation (R (2) = 0.6259; P = 0.0111).The threshold current also decreased with an increase in electrode impedance (R (2) = 0.2554; P = 0.0147)., Conclusions: Electrochemically treated porous platinum electrodes can stimulate localized retinal areas. The threshold current necessary to stimulate the retina was influenced by residual scleral thickness and the electrode tightness of fit against the sclera.

Purpose: To determine whether transcorneal electrical stimulation (TES) has neuroprotective effects on the photoreceptors, and whether it slows the rate of decrease of the electroretinogram (ERG) in rhodopsin P347L transgenic (Tg) rabbits., Methods: Six-week-old Tg rabbits received TES through a contact lens electrode on the left eye weekly for 6 weeks. The right eyes received sham stimulation on the same days. Electroretinograms (ERGs) were recorded before and at 12 weeks after the TES. After the last ERG recordings, the animals were euthanized for morphologic analysis of the retinas. Immunohistochemical (IHC) analysis was performed to detect the immunostaining by peanut agglutinin (PNA) and rhodopsin antibodies in the retinas., Results: The a- and b-wave amplitudes of the photopic ERGs and the b-wave amplitudes of the scotopic ERGs at higher stimulus intensities were significantly larger in the TES eyes than in the sham stimulated eyes (P<0.05, respectively). Morphologic analyses showed that the mean thickness of the outer nuclear layer (ONL) in the visual streak at 12 weeks was significantly thicker in TES eyes than in sham-stimulated eyes (P<0.05). IHC showed that the immunostaining by PNA and rhodopsin antibody in the TES-treated retinas was stronger than that in the sham-stimulated retinas., Conclusions: TES promotes the survival of photoreceptors and preserves the ERGs in Tg rabbits. Although further investigations are necessary before using TES on patients, these findings indicate that TES should be considered for therapeutic treatment for RP patients with a P347L mutation of rhodopsin.

Purpose: To examine the safety and effectiveness of a retinal prosthesis that is implanted semichronically in two patients with advanced retinitis pigmentosa (RP)., Methods: Two eyes of two patients with advanced RP had a retinal prosthesis implanted in a sclera pocket of one eye. The visual acuity of both eyes before the implantation was bare light perception. Phosphenes were elicited by suprachoroidal-transretinal stimulation (STS). The internal devices of the STS were implanted under the skin on the temporal side of the head, and the 49 electrode-array was implanted in the scleral pocket of one eye. Biphasic electrical pulses (duration, 0.5 ms; frequency, 20 Hz) were delivered through nine active electrodes. The threshold current was determined by currents ≤1 mA. Behavioral tasks were used to determine the functioning of the prosthesis., Results: The surgery was completed without a retinal detachment and retinal/vitreous hemorrhage. The implanted STS system remained functional for the 4-week test period. Phosphenes were elicited by currents delivered through six electrodes in Patient 1 and through four electrodes in Patient 2. The success of discriminating two bars was better than the chance level in both patients. In Patient 2, the success of a grasping task was better than the chance level, and the success rate of identifying a white bar on a touch panel increased with repeated testing., Conclusions: Semichronic implantation of a microelectrode-STS system showed that it was safe and remained functional for at least 4 weeks in two patients with advanced RP. (www.umin.ac.jp/ctr number, R000002690.).

Purpose: To compare retinal reflectance changes (RCs) elicited by transcorneal electrical stimulation (TES) to those elicited by electrical stimulation of the optic chiasma (OX)., Methods: Two eyes of two cats were studied under general anesthesia. Biphasic electrical pulses at 20 Hz were applied for 5 ms in TES, and monophasic pulses were applied for 50 μs at 100 Hz in OX stimulation. Fundus images observed with near-infrared light (800-880 nm) were recorded every 25 ms for 26 s, beginning 2 s before and ending 20 s after the electrical stimulation. To improve the signal-to-noise ratio, the images of ten consecutive recordings were averaged. Two-dimensional topographic maps of the RCs were constructed by subtracting images before the stimulation from those after the stimulation. The electrically evoked potentials (EEPs) were recorded at the OX. The effect of an intravitreal injection of tetrodotoxin (TTX) on the RCs elicited by electrical stimulation was also determined., Results: After electrical stimulation, RCs were observed at the optic disc, retinal arteries, and retinal veins. The two-dimensional maps of the RCs elicited by both TES and OX stimulation were similar. The latency of the RCs ranged from 2.0 to 4.0 s, and the peak occurred 6 to 9 s after the onset of the ES. The intensity of the RCs was correlated with the amplitude of EEP elicited by TES stimulation. The RCs disappeared after the TTX injection in both TES and OX stimulation., Conclusions: TES activates principally the retinal ganglion cells, and a change in the blood flow is initiated thereafter., (© Japanese Ophthalmological Society 2011.)

We developed a novel CMOS-based multichip flexible neural stimulator with on-chip stimulation generator. It enables simultaneous multi-site stimulation. We also propose a new type of multi-chip retinal stimulator with single electrode / unit chip configuration. We successfully performed simultaneous multi-site stimulation in an in vivo retinal stimulation experiment using a rabbit.

Purpose: To investigate the direct effect of electrical stimulation (ES) on the induction of brain-derived neurotrophic factor (BDNF) from cultured retinal Müller cells., Methods: Müller cells were isolated from rat retinas. ES was applied to passage 1 Müller cells with biphasic pulses (duration, 1 ms; frequency, 20 Hz; current, 10 mA) for 30 minutes. The changes in gene expression after ES were analyzed with microarrays. The mRNA and protein levels of BDNF were determined at each time point after ES by RT-PCR and ELISA, respectively. RT-PCR was also performed at 3 hours after ES of Müller cells that had been exposed to 1 microM nifedipine, a blocker of L-type voltage-dependent calcium channels (L-VDCCs)., Results: Microarray analyses showed an upregulation of 245 genes, including BDNF. The mRNA level of BDNF increased significantly (P < 0.05; by approximately 1.2-fold over that of the control) at 2 and 3 hours after ES. The intracellular protein level was upregulated significantly (by approximately 1.4-fold) at 6 hours after ES, whereas the extracellular level did not change at any time point. The total protein level of BDNF increased significantly ( approximately 1.3-fold) at 6 hours after ES. The increase in the mRNA level of BDNF was fully suppressed by exposure of the Müller cells to nifedipine., Conclusions: These results demonstrate that ES directly upregulates the transcriptional induction of BDNF through L-VDCCs in cultured Müller cells. The ES of Müller cells may be used to supply endogenous BDNF to the retina.

Purpose: To investigate the effect of electrical stimulation (ES) on the induction of insulin-like growth factor 1 (IGF-1) in cultured retinal Müller cells., Methods: Müller cells were isolated from rat retinas. ES was applied to Müller cells of passage 1 with biphasic pulses (duration, 1 ms; frequency, 20 Hz; current, 0-10 mA) for 30 min. The mRNA level of IGF-1 was determined by reverse transcription-polymerase chain reaction (RT-PCR) immediately to 2 h after ES. The change of intracellular calcium concentration ([Ca(2+)]) induced by ES was monitored by Ca(2+) imaging with Fura 2-AM. Ca(2+) imaging and RT-PCR were performed with and without the application of l muM nifedipine, an L-type calcium channel blocker., Results: The mRNA level of IGF-1 was increased significantly (P<0.05) by about 1.3-fold immediately after 10 mA ES. [Ca(2+)] began to increase immediately after the start of ES, reached a maximum of approximately 1.8-fold, and continued to increase until about 20 min after the ES. The inductions of IGF-1 transcription and Ca(2+) influx were suppressed by nifedipine., Conclusions: These results indicate that the enhancement of IGF-1 transcription by ES in cultured Müller cells depends largely on Ca(2+) influx through L-type Ca(2+) channels.

Multi-finger structure was proposed to improve flexibility of the CMOS LSI-based multi-chip retinal stimulator. A dual-finger retinal stimulator was fabricated and its functionality was demonstrated in retinal stimulation experiments on rabbit's retina, We also proposed an idea of pulsed-powering operation scheme for the multi-chip flexible retinal stimulator. We compared the pulsed-powering scheme with conventional one in a simulation, and show that the pulsed-powering can be an alternative operation scheme for the neural stimulator that provides an improved safety to the biological tissue.

The purpose of this study is to determine the threshold suprachoroidal-transretinal stimulation (STS) current that results in retinal damage in rabbits. Biphasic STS pulses (anodic first, frequency 20 Hz) were used to stimulate the retina of pigmented rabbits (n = 18) continuously for 1 h using a 100 microm diameter platinum wire electrode. The STS current that induced retinal damage after 1 h was determined by ophthalmoscopy or by fluorescein angiography (FA) independently. The effect of the pulse duration on the threshold current was investigated. Histological studies were performed after electrical stimulation experiments. The threshold for a safe current to the retina was 0.6 mA for a duration of 0.5 ms. The threshold for a safe charge increased approximately linearly with an increase of stimulus duration but the threshold for a safe current decreased logarithmically with an increase of duration. The threshold for a safe electrical energy remained almost constant for all durations. Histological examination showed severe retinal damage when the current exceeded the threshold, with more damage in the inner layers compared with the outer layers of the retina. The threshold for the safe current was higher than that reported for direct stimulation of neural tissues, suggesting that the STS method was safe and able to be used with a retinal prosthesis. Because the threshold for the safe charge was lower with shorter pulse durations, care should be taken using pulses of short durations.

We have performed in vivo electric stimulation experiments on rabbit retina to demonstrate feasibility of CMOS LSI-based multi-chip flexible neural stimulator for retinal prosthesis. We have developed new packaging structure with an improved flexibility and device control system which totally controls the LSI-based multi-chip stimulator, counter electrode, and stimulation generator. We have implanted the fabricated multi-chip stimulator into sclera pocket for STS (Suprachoroidal Transretinal Stimulation) configuration. We successfully obtained EEP (Electrically Evoked Potential) on visual cortex evoked by the multi-chip stimulator.

Background: To evaluate inner-retinal function by pupillary constrictions and phosphenes evoked by transcorneal electrical stimulation (TES) in patients with hereditary retinal degeneration., Methods: Consecutive 20 eyes of 20 patients (16 with retinitis pigmentosa (RP); and four with cone-rod dystrophy (CRD)) whose visual acuity was equal to or worse than 20/2000 at Osaka University Hospital and eight eyes of eight healthy subjects were enrolled. TES was performed on with a contact lens stimulating electrode. The electrically evoked pupillary response (EEPR) was recorded by a pupillometer, and the phosphenes by the subjective responses. Three electrical current thresholds were determined: T1, threshold current for initial phosphene; T2, threshold for eliciting a phosphene extending into the central field; and P, threshold for a relative pupillary constriction > or = 3%. The EEPR and phosphene thresholds were compared with the visual acuity or the visual field., Results: All T1, T2 and P were significantly higher in patients than in normals (Mann-Whitney, P<0.001). Both T1 and T2 were not correlated with visual acuity but depended on the area and location of the residual visual field. T1 and T2 in RP eyes with a EEPR was significantly lower than that in RP eyes without an EEPR. During TES, all subjects and patients had no pain, and no complications except for a slight corneal superficial punctuate keratopathy., Conclusions: The safety and the efficacy of TES to estimate the residual inner-retinal function in patients with retinal degeneration indicate that TES can be used as one of the most important test to select candidates for retinal prostheses.

Background: A new method of stimulating the retina electrically, called suprachoroidal transretinal stimulation (STS), was shown to be effective in eliciting electrically evoked cortical potentials (EEPs) in Royal College of Surgeons (RCS) rats. Before extending this technique to patients, it is important to determine its safety and feasibility in eliciting EEPs from medium-size animal (rabbits). The purpose of this study was to determine the safety and efficacy of the surgical procedures used to implant an multichannel electrode array into a scleral pocket, and to determine whether the implanted electrodes can stimulate the retina effectively., Methods: These acute experiments were conducted on six rabbits. An array of eight gold microelectrodes, embedded in polyimide, was implanted into a scleral pocket over the visual streak area. The size of the microarray was 2 x 4 x 0.180 mm. The reference electrode was implanted into the vitreous. The electrode array and reference electrodes were connected to a stimulator to deliver monophasic current pulses. Cortical responses were recorded with a stainless steel electrode implanted into each rabbit's skull over the visual cortex. After the experiment, the eyes and electrodes were examined histologically., Results: The surgical procedures for electrode implantation were accomplished without serious complications. EEPs were recorded after monophasic electrical pulse stimulation from each electrode. The mean threshold for EEPs was 55.0+/-10.0 microA with a 0.5-ms duration inward current pulse. The charge delivered at threshold was about 27.5 nC, and the charge density was about 56.0 microC/cm2. Histopathological examination of the retinal tissue around the area of stimulation did not show damage at the light microscope level with the electrical parameters used., Conclusions: Our technique for STS with an intrascleral microelectrode array is safe in rabbit eyes, and EEPs were elicited by current densities that did not induce tissue damage. These results suggest that STS via intrascleral multichannel electrodes is a feasible method for stimulating the retina.

Purpose: To determine whether wire microelectrodes implanted in the optic disc can be used to elicit cortical potentials., Methods: Two or four platinum wire electrodes of two types, viz., the cut-end type and the exposed-tip type, were inserted through the vitreous and fixed in the optic disc of 16 rabbit eyes. Electrically evoked potentials (EEPs) were recorded after bipolar electrical stimulation with the two wire electrodes and by different combinations of the four-electrode system. The optic discs were examined histologically after the experiment., Results: The wire electrodes were successfully implanted and fixed into different positions of the optic disc without serious complications in all 16 eyes. EEPs could be elicited after bipolar electrical stimulation of the optic nerve using either the two-electrode system or different pairs of the four-electrode system. Threshold charge densities to elicit EEPs were 0.32-0.64 mC/cm(2) in eyes using the cut-end type of electrodes and 0.93-6.21 muC/cm(2) in eyes using the exposed-tip type. The amplitude of the EEPs increased with increasing electrical stimulus intensities. Histological evaluation revealed limited damage to the neural tissue adjacent to the electrode track., Conclusions: The visual cortex can be activated by direct microelectrical stimulation of the optic nerve. The acute implantation of the wire microelectrodes into the optic disc by a transvitreal approach is feasible and results in only limited damage to the optic nerve.

Purpose: To investigate whether electrical stimulation of the optic nerve can elicit an electrical evoked potential (EEP) in rabbits and to determine whether such stimulation is a useful approach for the placement of a visual prosthesis., Methods: Two needle-type electrodes were inserted into the optic nerve using a transvitreal approach. For electrical stimulation, monophasic and biphasic pulses were used. By stimulating the optic nerve, the EEP was elicited. After evaluation of the EEP, a histological study was carried out., Results: When electrical stimulation was applied, the EEP could be recorded. The threshold with monophasic and biphasic stimulation was 10 +/- 0 microA and 20 +/- 8.2 microA, respectively. Histological examination revealed no major complications, such as bleeding or degeneration, which might have resulted from the insertion of electrodes or the electrical stimulation., Conclusions: Electrical stimulation of the optic nerve can elicit an EEP, suggesting that this approach may be useful for a visual prosthesis system.