In Vivo Photovoltaic Performance of a Silicon Nanowire Photodiode-Based Retinal Prosthesis.

Purpose:For more than 20 years, there has been an international, multidisciplinary effort to develop retinal prostheses to restore functional vision to patients blinded by retinal degeneration. We developed a novel subretinal prosthesis with 1512 optically addressed silicon nanowire photodiodes, which transduce incident light into an electrical stimulation of the remaining retinal circuitry. This study was conducted to evaluate the efficacy of optically driving the subretinal prosthesis to produce visual cortex activation via electrical stimulation of the retina. Methods:We measured electrically evoked potential responses (EEPs) in rabbit visual cortex in response to illumination of the subretinal nanowire prosthesis with pulsed 852-nm infrared (IR) light. We compared the EEP responses to visually evoked potential responses (VEPs) to pulsed 532-nm visible light (positive control) and pulsed 852-nm IR light (negative control). Results:Activating the devices with IR light produced EEP responses with a significantly higher trough-to-peak amplitude (54.17 ± 33.4 μV) than IR light alone (24.07 ± 22.1 μV) or background cortical activity (23.22 ± 17.2 μV). EEP latencies were significantly faster than focal VEP latencies. Focal VEPs produced significantly higher amplitudes (94.88 ± 43.3 μV) than EEPs. We also demonstrated how an electrode placed on the cornea can be used as a noninvasive method to monitor the function of the implant. Conclusions:These results show that subretinal electrical stimulation with nanowire electrodes can elicit EEPs in the visual cortex, providing evidence for the viability of a subretinal nanowire prosthetic approach for vision restoration

Temporal structure in spiking patterns of ganglion cells defines perceptual thresholds in rodents with subretinal prosthesis.

Subretinal prostheses are designed to restore sight in patients blinded by retinal degeneration using electrical stimulation of the inner retinal neurons. To relate retinal output to perception, we studied behavioral thresholds in blind rats with photovoltaic subretinal prostheses stimulated by full-field pulsed illumination at 20 Hz, and measured retinal ganglion cell (RGC) responses to similar stimuli ex-vivo. Behaviorally, rats exhibited startling response to changes in brightness, with an average contrast threshold of 12%, which could not be explained by changes in the average RGC spiking rate. However, RGCs exhibited millisecond-scale variations in spike timing, even when the average rate did not change significantly. At 12% temporal contrast, changes in firing patterns of prosthetic response were as significant as with 2.3% contrast steps in visible light stimulation of healthy retinas. This suggests that millisecond-scale changes in spiking patterns define perceptual thresholds of prosthetic vision. Response to the last pulse in the stimulation burst lasted longer than the steady-state response during the burst. This may be interpreted as an excitatory OFF response to prosthetic stimulation, and can explain behavioral response to decrease in illumination. Contrast enhancement of images prior to delivery to subretinal prosthesis can partially compensate for reduced contrast sensitivity of prosthetic vision

A Novel In Vitro Sensing Configuration for Retinal Physiology Analysis of a Sub-Retinal Prosthesis

This paper presents a novel sensing configuration for retinal physiology analysis, using two microelectrode arrays (MEAs). In order to investigate an optimized stimulation protocol for a sub-retinal prosthesis, retinal photoreceptor cells are stimulated, and the response of retinal ganglion cells is recorded in an in vitro environment. For photoreceptor cell stimulation, a polyimide-substrate MEA is developed, using the microelectromechanical systems (MEMS) technology. For ganglion cell response recording, a conventional glass-substrate MEA is utilized. This new sensing configuration is used to record the response of retinal ganglion cells with respect to three different stimulation methods (monopolar, bipolar, and dual-monopolar stimulation methods). Results show that the geometrical relation between the stimulation microelectrode locations and the response locations seems very low. The threshold charges of the bipolar stimulation and the monopolar stimulation are in the range of 10∼20 nC. The threshold charge of the dual-monopolar stimulation is not obvious. These results provide useful guidelines for developing a sub-retinal prosthesis

Photovoltaic restoration of sight with high visual acuity

Patients with retinal degeneration lose sight due to the gradual demise of photoreceptors. Electrical stimulation of surviving retinal neurons provides an alternative route for the delivery of visual information. We demonstrate that subretinal implants with 70-μm-wide photovoltaic pixels provide highly localized stimulation of retinal neurons in rats. The electrical receptive fields recorded in retinal ganglion cells were similar in size to the natural visual receptive fields. Similarly to normal vision, the retinal response to prosthetic stimulation exhibited flicker fusion at high frequencies, adaptation to static images and nonlinear spatial summation. In rats with retinal degeneration, these photovoltaic arrays elicited retinal responses with a spatial resolution of 64 ± 11 μm, corresponding to half of the normal visual acuity in healthy rats. The ease of implantation of these wireless and modular arrays, combined with their high resolution, opens the door to the functional restoration of sight in patients blinded by retinal degeneration

Biocompatibility of a Conjugated Polymer Retinal Prosthesis in the Domestic Pig

The progressive degeneration of retinal photoreceptors is one of the most significant causes of blindness in humans. Conjugated polymers represent an attractive solution to the field of retinal prostheses, and a multi-layer fully organic prosthesis implanted subretinally in dystrophic Royal College of Surgeons (RCS) rats was able to rescue visual functions. As a step toward human translation, we report here the fabrication and in vivo testing of a similar device engineered to adapt to the human-like size of the eye of the domestic pig, an excellent animal paradigm to test therapeutic strategies for photoreceptors degeneration. The active conjugated polymers were layered onto two distinct passive substrates, namely electro-spun silk fibroin (ESF) and polyethylene terephthalate (PET). Naive pigs were implanted subretinally with the active device in one eye, while the contralateral eye was sham implanted with substrate only. Retinal morphology and functionality were assessed before and after surgery by means of in vivo optical coherence tomography and full-field electroretinogram (ff-ERG) analysis. After the sacrifice, the retina morphology and inflammatory markers were analyzed by immunohistochemistry of the excised retinas. Surprisingly, ESF-based prostheses caused a proliferative vitreoretinopathy with disappearance of the ff-ERG b-wave in the implanted eyes. In contrast, PET-based active devices did not evoke significant inflammatory responses. As expected, the subretinal implantation of both PET only and the PET-based prosthesis locally decreased the thickness of the outer nuclear layer due to local photoreceptor loss. However, while the implantation of the PET only substrate decreased the ff-ERG b-wave amplitude with respect to the pre-implant ERG, the eyes implanted with the active device fully preserved the ERG responses, indicating an active compensation of the surgery-induced photoreceptor loss. Our findings highlight the possibility of developing a new generation of conjugated polymer/PET-based prosthetic devices that are highly biocompatible and potentially suitable for subretinal implantation in patients suffering from degenerative blindnes

Therapeutic Challenges to Retinitis Pigmentosa: From Neuroprotection to Gene Therapy

Syndromic retinitis pigmentosa (RP) is the result of several mutations expressed in rod photoreceptors, over 40 of which have so far been identified. Enormous efforts are being made to relate the advances in unraveling the patho-physiological mechanisms to therapeutic approaches in animal models, and eventually in clinical trials on humans. This review summarizes briefly the current clinical management of RP and focuses on the new exciting treatment possibilities. To date, there is no approved therapy able to stop the evolution of RP or restore vision. The current management includes an attempt at slowing down the degenerative process by vitamin supplementation, trying to treat ocular complications and to provide psychological support to blind patients. Novel therapeutic may be tailored dependant on the stage of the disease and can be divided in three groups. In the early stages, when there are surviving photoreceptors, the first approach would be to try to halt the degeneration by correction of the underlying biochemical abnormality in the visual cycle using gene therapy or pharmacological treatment. A second approach aims to cope with photoreceptor cell death using neurotrophic growth factors or anti-apoptotic factors, reducing the production of retino-toxic molecules, and limiting oxidative damage. In advanced stages, when there are few or no functional photoreceptors, strategies that may benefit include retinal transplantation, electronic retinal implants or a newly described optogenetic technique using a light-activated channel to genetically resensitize remnant cone-photoreceptor cells

Artificial vision: feasibility of an episcleral retinal prosthesis & implications of neuroplasticity

Background. A visual prosthesis is a conceptual device designed to activate residual functional neurons in the visual pathway of blind individuals to produce artificial vision. Such device, when applied to stimulate the vitreous surface of the retina, has proven feasible in producing patterned light perception in blind individuals suffering from dystrophic diseases of the retina, such as aged-related macular degeneration (AMD). However the practicality of such approach has been challenged by the difficulty of surgical access and the risks of damaging the neuroretina. Positioning a visual implant over the scleral surface of the eye could present a safer alternative but this stimulation modality has not been tested in diseased retinas. Additionally, recent research has shown that the adult neocortex retains substantial plasticity following a disruption to its visual input and the potential deterioration in visual capabilities as a result of such experience modification may undermine the overall bionic rescue strategy. Methods. Two animal models mimicking the principal pathologies found in AMD, namely photoreceptor degeneration and reduced retinal ganglion cell mass, were used to evaluate the efficacy of trans-scleral stimulation of the retina by recording electrical evoked potentials in the visual cortex. The visual performance following the loss of pattern vision induced by bilateral eyelid suturing in adult mice was examined by analysing visual evoked potentials. Findings. Spatially differentiated cortical activations were obtained notwithstanding the underlying retinopathy in the experiment animals. The charge density thresholds were found to be similar to controls and below the bioelectric safety limit. After prolonged visual deprivation (weeks) in the mouse, the visual cortical responses evoked by either electrical or photic stimuli were both significantly reduced. An assessment of different visual capabilities using patterned stimuli demonstrated that whilst visual acuity and motion sensitivity were preserved, significant depression in luminance and contrast sensitivities was detected. Conclusion. Trans-scleral stimulation of the retina is a feasible approach for the development of a visual prosthesis. Following visual loss the adult brain exhibits significant experience-dependent modifications. These new insights may force a revision on the current bionic rescue strategy

Retinal ganglion cells : physiology and prosthesis

The retina is responsible for encoding different aspects of the visual world. Light enters the eyes and is converted by the photoreceptors into electrochemical signals. These signals are processed by the retinal network and proceed afferently to the brain via the axons of the retinal ganglion cells (RGCs). The RGCs outputs are in the form of action potentials (spikes), which encrypt the visual information in terms of spike shape, firing frequencies, and the firing patterns. When the photoreceptors are gone due to disease, vision is lost. The idea of a retinal prosthesis is to activate the surviving RGCs by electrical stimulation in order to recreate vision. In this thesis, I have studied the physiological properties of the RGCs, and reconstructed natural RGC spike trains by electrical stimulation. Chapter 1 introduces the anatomy of the retina and the retinal neurons. How the RGCs respond to light. Electrical stimulation is also discussed. A brief historical summary of the receptive field properties and cell physiology is also presented. Chapter 2 characterizes the intrinsic properties of 16 morphologically defined types of rat RGCs. The intrinsic properties include the biophysical properties due to morphology and dendritic stratification, in addition to physiological properties such as firing behaviours. These properties are also compared with the cat RGC intrinsic properties in order to investigate the variations between the morphologically similar RGCs of the two species. The results suggest that the RGCs among species, even with similar morphologies, do not have conservative intrinsic properties. Chapter 3 examines the details of the spiking properties of the different rat RGC types. Spikes are initiated at the axonal initial segment. A 'single' spike recorded at the soma consists of an axonal spike and a somatic spike. The existence of the two spikes can be recognized by two humps in the phase plot, and further revealed in the higher derivatives of the membrane potential. A principal component analysis shows that the parameters extracted from the phase plots are very useful for a model-independent rat RGC classification. Chapter 4 establishes the foundations for electrical stimulation of the retina. The question is to what extent optimum placement of the stimulating and reference electrodes might be affected by anatomical location. Here we placed the stimulating electrode above or below the retinal inner limiting membrane and found no statistical difference between the thresholds. In addition, reflective axonal spikes from the cut end are discussed. Chapter 5 combines the knowledge obtained in the previous chapters for the sole purpose of reproducing natural RGC outputs when using electrical stimulation. The light responses of the eye under saccadic movements were recorded and used to form the stimulus patterns. The reconstructions were performed on the brisk-transient (BT) and the brisk-sustained (BS) RGCs. Our results suggested that BT RGCs are more capable of following the stimulated stimulus patterns over a wide range of frequencies than the BS RGCs. Chapter 6 concludes the whole thesis

Optimal electrical activation of retinal ganglion cells

Retinal prostheses are emerging as a viable therapy option for those blinded by degenerative eye conditions that destroy the photoreceptors of the retina but spare the retinal ganglion cells (RGCs). My research sought to address the issue of how a retinal prosthesis might best activate these cells by way of electrical stimulation. Whole-cell patch clamp recordings were made in explanted retinal wholemount preparations from normally-sighted rats. Stimulating electrodes were fabricated from nitrogen-doped ultra-nanocrystalline diamond (N-UNCD) and placed on the epiretinal surface, adjacent to the cell soma. Electrical stimuli were delivered against a distant monopolar return electrode. Using rectangular, biphasic constant current waveforms as employed by modern retinal prostheses, I examined which waveform parameters had the greatest effect on RGC activation thresholds. In a second set of experiments intracellular current injection was employed to assess the effectiveness of sinusoidal current waveforms in selectively activating different RGC subsets. These recordings were also used to validate a biophysical model of RGC activation. Where possible, recorded cells were identified and classified based on 3D confocal reconstruction of their morphology. Electrodes fabricated from N-UNCD were able to electrically activate RGCs while remaining well within the electrochemical limits of the material. They were found to exhibit high electrochemical stability and were resistant to morphological and electrochemical changes over one week of continuous pulsing at charge injection limits. Retinal ganglion cells invariably favoured cathodic-first biphasic current pulses of short first-phase duration, with a small interphase interval. The majority of cells (63\%) were most sensitive to a highly asymmetric waveform: a short-cathodic phase followed by a longer duration, lower amplitude anodic phase. Using the optimal interphase interval led to median charge savings of 14\% compared to the charge required in the absence of any inter-phase interval. Optimising phase duration resulted in median charge savings of 22\%. All RGCs became desensitised to repetitive electrical stimulation. The efficacy of a given stimulus delivered repeatedly decreased after the first stimulus, stabilising at a lower efficacy by the thirtieth pulse. This asymptotic efficacy decreased with increasing stimulus frequency. Cells with smaller somas and dendritic fields were better able to sustain repetitive activation at high frequency. Intracellular sinusoidal stimulation was used to demonstrate that certain RGC subsets, defined on the basis of morphological type, stratification, and size, were more responsive to high frequency stimulation. Simulated RGC responses were validated by experimental data, which confirmed that ON cell responses were heavily suppressed by stimulus frequencies of 20 Hz and higher. OFF cells, on the other hand, were able to sustain repetitive activation over all tested frequencies. Additional simulations suggest this difference may be plausibly attributed to the presence of low-voltage-activated calcium channels in OFF but not ON RGCs. The results of my work demonstrate that (a) N-UNCD is a suitable material for retinal prosthesis applications; (b) a careful choice of electrical waveform parameters can significantly improve prosthesis efficacy; and (c) it may be possible to bias neural activation for certain RGC populations by varying the frequency of stimulation

Zapping the Retina - Understanding electrical responsiveness and electrical desensitization in mouse retinal ganglion cells

The field of science and technology has come a long way since the famous 70’s science fiction series “The Six Million Dollar Man,” where a disabled pilot was transformed into a bionic superhero after receiving artificial implants. What was indeed once a science fiction has now turned into a science fact with the development of various electronic devices interfacing the human neurons in the brain, retina, and limbs. One such advancement was the development of retinal implants. Over the past two decades, the field of retinal prosthetics has made significant advancement in restoring functional vision in patients blinded by diseases such as Retinitis pigmentosa (RP) and Age-related macular degeneration (AMD). RP and AMD are the two leading cause of degenerative blindness. While there is still no definitive cure for either of these diseases, various treatment strategies are currently being explored. Of the various options, the most successful one has been the retinal implants. Retinal implants are small microelectrode or photodiode arrays, which are implanted in the eye of a patient, to stimulate the degenerating retina electrically. They are broadly classified into three types depending on the placement ̶ epiretinal (close proximity to retinal ganglion cells, RGCs) , subretinal (close proximity to bipolar cells, BP) and suprachoroidal (close proximity to choroid). While the ongoing human trials have shown promising results, there remains a considerable variability among patients concerning the quality of visual percepts which limits the working potential of these implants. One such limitation often reported by the implanted patients is “ fading” of visual percepts. Fading refers to the limited ability to elicit temporally stable visual percepts. While, this is not a primary concern for epiretinal implants , it is often observed in subretinal and suprachoroidal implants which use the remaining retinal network to control the temporal spiking pattern of the ganglion cells. The neural correlate of fading is often referred to as “electrical desensitization”, which is the reduction of ganglion cell responses to repetitive electrical stimulation . While much is known about the temporal component of desensitization ( time constant, τ), the spatial aspects (space constant, λ) has not been well characterized. Further, how both these aspects interact to generate spiking responses, remains poorly understood. These crucial questions formed the critical components of my thesis. To address these questions, we stimulated the retinal network electrically, with voltage and current pulses and recorded the corresponding spiking activity using the microelectrode arrays (MEAs). While addressing the primary question of my thesis, we were able to address few idiosyncrasies which has currently stymied the field of retinal prosthetics. At a conceptual level, we have developed an experimental and analysis framework by which one can identify the single stimulus that will activate the most ganglion cells (Chapter 2, Part 1). This stimulus is optimal for ‘blind’ experiments where the specific response properties of each cell are unknown. Furthermore, we attempted to understand the correspondence between the electrical response patterns and visual response types (Chapter 2, Part2). In Chapter 3, we sought to understand better how the visual responses parameters change during ongoing electrical stimulation. We demonstrated that apart from the adaptation occurring due to visual stimulation and invitro experimental conditions, the electrical stimulation alters the RGC visual responses, suggesting the requirement for stimulation-induced changes to be incorporated in the designing of stimulation paradigms for the implant. Finally addressing the primary question (Chapter 4) of my thesis with which we started, we were able to demonstrate, that the electrical desensitization requires the interaction of both time and distance and is not a global phenomenon of the retina. In the final chapter (Chapter 5) we summarize the results of the thesis, discuss the key outcomes and its relevance to the prosthetic field and other vision restoration strategies and the potential future directions of this research. Therefore, in future, to improve the efficacy of retinal prostheses and patient outcomes, it is crucial to have an in-depth understanding of the responsiveness of the retinal circuitry to electrical stimulation