Array resolution

For rat eye implantations, we produced arrays of 0.8 × 1.2 mm in size, containing 13, 45 and 186 pixels of 280 μm, 140 μm and 70 μm in width, respectively (Fig. 1). A local return electrode in each pixel helps confine electric current in front of each pixel, an essential feature for high-resolution stimulation. High-charge injection is achieved using iridium oxide coating of the electrodes21. Light reflection from the device is minimized using an antireflection coating consisting of 60 nm of SiO 2 and 70 nm of SiN x . The pixel density with 70 μm pixels is 209 pixels per mm2, corresponding to more than 4,800 pixels over an area similar to the ARGUS II implant of Second Sight Inc. (5 × 5 mm).

Anatomy of subretinal implantation

Eight arrays with pixel sizes of 280 μm, 9 with 140 μm and 10 with 70 μm pixels were successfully implanted subretinally in 27 rats (see Methods): 12 wild-type (Long-Evans) and 15 animals with retinal degeneration (RCS), as listed in Table 1. Follow-up with optical coherence tomography (OCT) imaging revealed gradual resolution of mild retinal oedema or retinal detachment within the first few weeks after implantation. Figure 2c depicts a typical OCT image of an implant in the subretinal space. Because of the much higher refractive index of silicon (n≈3.6) than that of ocular tissues (n≈1.4), the implant appears almost three times thicker than it actually is: 77 μm instead of 30 μm. The upper surface of the implant is in close proximity to the INL, where the target neurons for electrical stimulation (bipolar cells) reside. Fluorescein angiography revealed normal vasculature and good perfusion of the retina overlying the implants (Fig. 2b). As the Si array blocks visible light, there is no fluorescent background from the choroid, which improves visibility of the retinal vasculature above the implant. As expected, the presence of the implant between the retinal pigmented epithelium (RPE) and photoreceptors in wild-type (WT) animals caused gradual degradation and disappearance of the photoreceptors above the implant while leaving the adjacent retina unaffected (Fig. 2c). In 7 out of 12 cases of WT rats, we found fluorescent deposits above the implant, beginning a few weeks following the implantation. These are likely associated with degeneration of photoreceptors detached from the RPE by the implant. In RCS rats, diffuse autofluorescence was distributed over the whole retina, as part of the degenerative process24. There were no cases of infection or inflammatory reaction during the 6 months of follow-up.

Table 1 The range of stimulation thresholds for different pixel sizes. Full size table

Figure 2: Photodiode arrays in the eye. (a) Scanning electron micrograph of a photodiode array with 70 μm pixels above the RPE in a porcine eye. Scale bar, 70 μm. (b) Fluorescein angiography 10 days after subretinal implantation of the photodiode array in WT rats shows good perfusion over the implant with no staining or leakage. Scale bar, 500 μm. (c) OCT of a WT rat 12 weeks after subretinal implantation. INL (pointed by the arrow) is in close proximity to the implant, with no evidence of retinal oedema or injury. Photoreceptor layer above the implant is missing due to prolonged separation from the RPE. Implant appears 77 μm thick instead of actual 30 μm due to much higher refractive index of silicon (n≈3.6) than that of ocular tissues (n≈1.4). (d) OCT of an RCS rat 7 days after implantation. INL (indicated by the arrow) is in close proximity to the implant. As expected, due to retinal degeneration, there is no photoreceptor layer. Full size image

To assess potential effect of the implant on neural retina, the thickness of the inner retinal layers (from the INL up to the inner limiting membrane (ILM)) was measured in 9 WT rats over the follow-up period using OCT. No significant change in thickness of the inner retina has been observed: average thickness above the implant changed from 58 μm during the first month after implantation to 54 μm at the end of the follow-up period (matched t-test, P=0.4). Average inner retinal thickness away from the implant decreased from 64 to 58 μm, but the difference also was not statistically significant (matched t-test P=0.2). In a few animals, we observed gradual retinal thinning or decrease in vascularization along one of the longer edges of the implant. This effect is probably induced by elevation of the middle part of the rectangular implant above the RPE in a spherically shaped eyeball. This may not be a problem in human eyes, as the curvature of the eyeball is about five times larger, and this issue can be avoided using arrays of a round shape rather than rectangular.

Visual-evoked potentials

Following successful subretinal implantation, trans-cranial screw electrodes were placed over the primary visual cortex (see Methods) and animals were observed for another week before recording VEP signals. Three experiments were performed as controls. First, to check for natural sensitivity of the rat retina to infrared light, near-IR (NIR) pulses were applied to the same spot size on the retina away from the implant at maximum irradiance and pulse duration: 20 mW mm−2 and 10 ms. No VEP responses were observed. Second, to check for possible direct effect of the stimulation current on the brain, retrobulbar injection of Lidocain 2% was applied to block signal transduction via optic nerve. This resulted in complete disappearance of VEP signals for both, the visible light and NIR. Third, NIR stimulation was applied to two deceased rats (which showed robust eVEP when alive), and it elicited no VEP, just a short stimulation artifact, similar to the one depicted in Fig. 3c (*).

Figure 3: Cortical responses in WT (normally sighted) animals. (a) VEP of the WT animal in response to white light at various luminance levels provided by dome stimulator. With brighter stimuli, the VEP amplitude increases and latency decreases. (b) NIR stimuli over the implant with 70 μm pixels elicited a prompt eVEP with similar structure but significantly shorter latencies: N1 and P2 at 19 ms and 53 ms, respectively. Amplitude of both N1 and P2 increased at higher irradiances. This recording was performed 4 months after implantation. (c) VEP (green) elicited by visible (635 nm) light projected onto 1 mm spot away from the implant and eVEP (blue and red) were recorded in the same WT animal implanted with 280 μm pixels array. eVEP were recorded in response to 4 ms NIR stimuli at peak irradiances of 2.5 (blue) and 10 mW mm−2 (red), repeated at 2 Hz. A short artifact caused by the photovoltaic current is indicated by *. Full size image

Typical eVEP response to photovoltaic stimulation was characterized by two early negative components at 15–21 ms (N1) and 35–41 ms (N2), followed by two positive peaks at 30–35 ms (P1) and 55–60 ms (P2). A later smaller positive peak (P3) typically had latency>100 ms (Fig. 3b). VEP response to the natural photopic stimuli was first obtained with the full-field uniform white illumination using the dome (Fig. 3a), and then with a 1 mm spot (same size as the implant) of red (635 nm) light projected from the slit lamp away from the implant. With both, the full-field and the spot illumination, the VEP had generally similar structure (N1, P1, N2, P2, P3), but was delayed compared with the response to photovoltaic stimulation (Fig. 3c). Though the latency of N1 decreased with increased irradiance (Fig. 5d), the shortest latency observed was 40 ms—about 20 ms later than the first negative peak in photovoltaic stimulation. This difference is likely due to the fact that electrical stimulation elicits responses from the inner retinal neurons, bypassing the phototransduction process in the retina, which can take up to several tens of milliseconds25. It should be noted that much brighter light at 635 nm was required to elicit VEP responses equivalent in amplitude to those from the full-field white-light illumination (dome). This difference is likely to be due to at least two factors: much lower sensitivity of rodent retina at 635 nm as compared with white light26, and smaller area of the illuminated retina. It is quite possible that red light scattered from the small illuminated spot activated the whole retina in the eye.

The eVEP recorded from RCS rats had similar structure to that of the WT rats, as illustrated in Fig. 4b for RCS rat implanted with a 70-μm pixels array. However, the VEP in response to white light in RCS rats was very weak and slow, with no significant N1, even with very bright full-field stimuli (Fig. 4a). Visible light stimulation with 1 mm spot did not elicit any detectable VEP.

Figure 4: Cortical responses in animals with degenerate retina. VEP (a) and eVEP (b) recording from an RCS rat (age 17 weeks) implanted with 70 μm pixel-size array. (a) VEP traces in response to full-field (dome) white light stimuli at two irradiance levels show slow and weak response even at high illumination. N1 latency is 70 ms and P wave more than 200 ms (compared with faster and stronger response in WT rat, shown in Fig. 3a). (b) eVEP in the same rat in response to NIR stimulation over the implant at 10 mW mm−2, with 1 and 10 ms pulses. Longer pulse duration elicited stronger eVEP response. Full size image

To evaluate whether stimulation of the RGCs by subretinal photovoltaic array is mediated by the inner retinal neurons, a cocktail of synaptic transmission blockers was injected intravitreally in four WT rats. Such injections resulted in immediate disappearance of the cortical responses to both, the visible and NIR light (10 ms, up to 10 mW mm−2, Supplementary Fig. S2a), and both responses returned to normal the next day. This result suggests involvement of the inner retinal neurons and retinal network in subretinal stimulation. To control for potential effects of the intraocular pressure rise, intravitreal injections of a similar volume of fluid without the active ingredients have been performed, and they did not affect cortical responses (Supplementary Fig. S2b).

The normalized VEP amplitude increased with peak irradiance over four orders of magnitude of the visible light brightness, following a characteristic sigmoid curve (Fig. 5c). Over the central two orders of magnitude (0.05–2.5 cd m−2), the signal increased logarithmically. The eVEP response also increased logarithmically with peak irradiance over two orders of magnitude of the NIR irradiance (Fig. 5a), with no significant difference between the animal types. However, while N1 latency in VEP elicited by visible light significantly decreased with increased illumination (Fig. 5d); in eVEP elicited by NIR, the N1 latency did not vary with irradiance (Fig. 5b). Normalized eVEP could also be modulated by pulse duration while keeping the irradiance constant (Fig. 6a) and showed a logarithmic increase with pulse duration from 1 to 10 ms. In RCS group, the rise with pulse duration (Fig. 6a, red line) was slower than in the WT animals (Fig. 6a, blue line). Similarly, VEP elicited by visible light in WT animals increased in amplitude logarithmically with pulse duration (Fig. 6b).

Figure 5: VEP amplitude and latency as a function of peak irradiance for NIR and visible stimuli. (a) The eVEP amplitude (N1–P2), normalized to response at 10 mW mm−2, and averaged for 7 WT and 7 RCS rats is plotted as a function of peak irradiance. Pulse duration was held constant at 10 ms. The eVEP amplitude increased logarithmically with peak irradiance, with no significant difference between the two animal types. (b) N1 latency in the same eVEP recordings did not vary with irradiance, and was very similar in the two animal groups. (c) Normalized VEP amplitude in WT rats, in response to white light full-field stimuli, averaged over six animals, and plotted as a function of the peak irradiance. For averaging between the animals, the VEP amplitude was normalized to the response at maximum irradiance. Error bars represent the s.e.m. (d) N1 latency as a function of the peak irradiance in the same data set. Full size image

Figure 6: VEP amplitude as a function of pulse duration. (a) eVEP amplitude (N1–P2), normalized to response at 10 ms, and averaged for 6 WT and 4 RCS rats. eVEP amplitude increased logarithmically with pulse duration from 1 to 10 ms, and slightly decreased at 20 ms. In the RCS group, the rise with pulse duration (red line) was smaller than in the WT animals (blue line). (b) Average VEP amplitude elicited by a 1 mm spot of visible light (635 nm), in 6 WT animals, as a function of pulse duration, normalized to its maximum value. Error bars represent the s.e.m., dashed lines represent logarithmic fit. Full size image

Stimulation thresholds were determined as the minimum stimuli peak irradiance eliciting a VEP signal with amplitude 3 s.d. above the noise level (see detailed description in Methods). Table 1 lists the average values and the range of stimulation thresholds for various pixel sizes with 4- and 10-ms pulse durations. The lowest threshold in this series was 0.25 mW mm−2 at 4 ms, observed in a WT animal implanted with 140 μm pixel array. On an average, the thresholds increased with decreasing pixel size: from 0.43 mW mm−2 with the 280 μm pixels to 1 mW mm−2 with the 140 μm arrays, and to 2.1 mW mm−2 with the implants having 70 μm pixels. Thresholds decreased, on average, by a factor of 1.8 when pulse duration increased from 4 to 10 ms. There was no statistically significant difference between the stimulation thresholds in RCS and in WT rats. As can be seen from comparison of the Figs 3 and 4, the shape and amplitude of the eVEP in RCS rats was quite similar to those of the WT responses.

Simultaneous pulsed stimulation of the retina with patterns representing visual scenes is more similar to stroboscopic vision rather than to the natural continuous illumination. At sufficiently high frequencies, the pulsed stroboscopic representation fuses into a continuous perception27. We assessed potential differences in frequency response to pulsed electrical and visible light stimuli by measuring the amplitude of VEP (N1 to P2) at various pulse-repetition rates (Fig. 7a). VEP amplitude, in response to electrical stimulation, decreased with increasing frequency from 2 to 20 Hz—similar to the visible light-induced response (Fig. 7b). From 20 to 40 Hz, the visible response continued to decrease, whereas the eVEP did not change as much. The difference becomes more profound taking into account the decrease of the photovoltaic current with frequency, as measured by the amplitude of the stimulation artifact (Fig. 7b). As expected from the RC circuit with a resistance-limited discharge rate, photovoltaic current decreased exponentially with frequency21.

Figure 7: VEP elicited by 1 mm visible light spot on the retina. (a) Robust VEP response to 5 (upper trace) and 10 Hz (lower trace) NIR stimulation in a WT rat. (b) Amplitude of VEP in response to NIR (red, averaged for n=18) and visible (green, averaged for n=6) stimuli at frequencies of 2–40 Hz. Error bars represent s.e.m. Blue dashed line depicts the frequency response of the photocurrent from the implant. Full size image

Electrophysiological responses of the WT and RCS rats remained very robust during the whole 6-month-long follow-up period. For example, the VEP responses shown in Fig. 3b were recorded 4 months after implantation. However, on average, the stimulation thresholds with 10 ms pulses increased by 58% over the follow-up period. As these implants were not protected by SiC coating for long-term stability, erosion of the silicon nitride antireflection coating of the chips23 in the body was expected to start affecting their performance over several months of implantation. Increase in the stimulation thresholds could also be attributed to retinal changes or to corneal oedema developed over time in some animals due to irritation by the gel and contact lens.