Corrigendum: Electrically-evoked responses for retinal prostheses are differentially altered depending on ganglion cell types in outer retinal neurodegeneration caused by Crb1 gene mutation.

[This corrects the article DOI: 10.3389/fncel.2023.1115703.].

Localization of hyperpolarization-activated cyclic nucleotide-gated channels in the vertebrate retinas across species and their physiological roles.

Transmembrane proteins known as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control the movement of Na+ and K+ ions across cellular membranes. HCN channels are known to be involved in crucial physiological functions in regulating neuronal excitability and rhythmicity, and pacemaker activity in the heart. Although HCN channels have been relatively well investigated in the brain, their distribution and function in the retina have received less attention, remaining their physiological roles to be comprehensively understood. Also, because recent studies reported HCN channels have been somewhat linked with the dysfunction of photoreceptors which are affected by retinal diseases, investigating HCN channels in the retina may offer valuable insights into disease mechanisms and potentially contribute to identifying novel therapeutic targets for retinal degenerative disorders. This paper endeavors to summarize the existing literature on the distribution and function of HCN channels reported in the vertebrate retinas of various species and discuss the potential implications for the treatment of retinal diseases. Then, we recapitulate current knowledge regarding the function and regulation of HCN channels, as well as their relevance to various neurological disorders.

Effective protection of photoreceptors using an inflammation-responsive hydrogel to attenuate outer retinal degeneration.

Retinitis pigmentosa (RP) is an outer retinal degenerative disease that can lead to photoreceptor cell death and profound vision loss. Although effective regulation of intraretinal inflammation can slow down the progression of the disease, an efficient anti-inflammatory treatment strategy is still lacking. This study reports the fabrication of a hyaluronic acid-based inflammation-responsive hydrogel (IRH) and its epigenetic regulation effects on retinal degeneration. The injectable IRH was designed to respond to cathepsin overexpression in an inflammatory environment. The epigenetic drug, the enhancer of zeste homolog 2 (EZH2) inhibitors, was loaded into the hydrogel to attenuate inflammatory factors. On-demand anti-inflammatory effects of microglia cells via the drug-loaded IRH were verified in vitro and in vivo retinal degeneration 10 (rd10) mice model. Therefore, our IRH not only reduced intraretinal inflammation but also protected photoreceptors morphologically and functionally. Our results suggest the IRH reported here can be used to considerably delay vision loss caused by RP.

Effects on Retinal Stimulation of the Geometry and the Insertion Location of Penetrating Electrodes.

Retinal implants have been developed and implanted to restore vision from outer retinal degeneration, but their performance is still limited due to the poor spatial resolution. To improve the localization of stimulation, microelectrodes in various three-dimensional (3D) shapes have been investigated. In particular, computational simulation is crucial for optimizing the performance of a novel microelectrode design before actual fabrication. However, most previous studies have assumed a uniform conductivity for the entire retina without testing the effect of electrodes placement in different layers. In this study, we used the finite element method to simulate electric fields created by 3D microelectrodes of three different designs in a retina model with a stratified conductivity profile. The three electrode designs included two conventional shapes - a conical electrode (CE) and a pillar electrode (PE); we also proposed a novel structure of pillar electrode with an insulating wall (PEIW). A quantitative comparison of these designs shows the PEIW generates a stronger and more confined electric field with the same current injection, which is preferred for high-resolution retinal prostheses. Moreover, our results demonstrate both the magnitude and the shape of potential distribution generated by a penetrating electrode depend not only on the geometry, but also substantially on the insertion depth of the electrode. Although epiretinal insertions are mainly discussed, we also compared results for subretinal insertions. The results provide valuable insights for improving the spatial resolution of retinal implants using 3D penetrating microelectrodes and highlight the importance of considering the heterogeneity of conductivities in the retina.

A novel convex acoustic lens-attached ultrasound drug delivery system and its testing in a murine melanoma subcutaneous model.

Target-specific drug release is indispensable to improve chemotherapeutic efficacy as it enhances drug uptake and penetration into tumors. Sono-responsive drug-loaded nano-/micro-particles are a promising solution for achieving target specificity by exposing them to ultrasound near tumors. However, the complicated synthetic processes and limited ultrasound (US) exposure conditions, such as limited control of ultrasound focal depth and acoustic power, prevent the practical application of this approach in clinical practice. Here, we propose a convex acoustic lens-attached US (CALUS) as a simple, economic, and efficient alternative of focused US for drug delivery system (DDS) application. The CALUS was characterized both numerically and experimentally using a hydrophone. In vitro, microbubbles (MBs) inside microfluidic channels were destroyed using the CALUS with various acoustic parameters (acoustic pressure [P], pulse repetition frequency [PRF], and duty cycle) and flow velocity. In vivo, tumor inhibition was evaluated using melanoma-bearing mice by characterizing tumor growth rate, animal weight, and intratumoral drug concentration with/without CALUS DDS. US beams were measured to be efficiently converged by CALUS, which was consistent with our simulation results. The acoustic parameters were optimized through the CALUS-induced MB destruction test (P = 2.34 MPa, PRF = 100 kHz, and duty cycle = 9%); this optimal parameter combination successfully induced MB destruction inside the microfluidic channel with an average flow velocity of up to 9.6 cm/s. The CALUS also enhanced the therapeutic effects of an antitumor drug (doxorubicin) in vivo in a murine melanoma model. The combination of the doxorubicin and the CALUS inhibited tumor growth by âˆ¼ 55% more than doxorubicin alone, clearly indicating synergistic antitumor efficacy. Our tumor growth inhibition performance was better than other methods based on drug carriers, even without a time-consuming and complicated chemical synthesis process. This result suggests that our novel, simple, economic, and efficient target-specific DDS may offer a transition from preclinical research to clinical trials and a potential treatment approach for patient-centered healthcare.

Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

Electrically-evoked responses for retinal prostheses are differentially altered depending on ganglion cell types in outer retinal neurodegeneration caused by Crb1 gene mutation.

Background: Microelectronic prostheses for artificial vision stimulate neurons surviving outer retinal neurodegeneration such as retinitis pigmentosa (RP). Yet, the quality of prosthetic vision substantially varies across subjects, maybe due to different levels of retinal degeneration and/or distinct genotypes. Although the RP genotypes are remarkably diverse, prosthetic studies have primarily used retinal degeneration (rd) 1 and 10 mice, which both have Pde6b gene mutation. Here, we report the electric responses arising in retinal ganglion cells (RGCs) of the rd8 mouse model which has Crb1 mutation. Methods: We first investigated age-dependent histological changes of wild-type (wt), rd8, and rd10 mice retinas by H&E staining. Then, we used cell-attached patch clamping to record spiking responses of ON, OFF and direction selective (DS) types of RGCs to a 4-ms-long electric pulse. The electric responses of rd8 RGCs were analyzed in comparison with those of wt RGCs in terms of individual RGC spiking patterns, populational characteristics, and spiking consistency across trials. Results: In the histological examination, the rd8 mice showed partial retinal foldings, but the outer nuclear layer thicknesses remained comparable to those of the wt mice, indicating the early-stage of RP. Although spiking patterns of each RGC type seemed similar to those of the wt retinas, correlation levels between electric vs. light response features were different across the two mouse models. For example, in comparisons between light vs. electric response magnitudes, ON/OFF RGCs of the rd8 mice showed the same/opposite correlation polarity with those of wt mice, respectively. Also, the electric response spike counts of DS RGCs in the rd8 retinas showed a positive correlation with their direction selectivity indices (r = 0.40), while those of the wt retinas were negatively correlated (r = -0.90). Lastly, the spiking timing consistencies of late responses were largely decreased in both ON and OFF RGCs in the rd8 than the wt retinas, whereas no significant difference was found across DS RGCs of the two models. Conclusion: Our results indicate the electric response features are altered depending on RGC types even from the early-stage RP caused by Crb1 mutation. Given the various degeneration patterns depending on mutation genes, our study suggests the importance of both genotype- and RGC type-dependent analyses for retinal prosthetic research.

Short pulses of epiretinal prostheses evoke network-mediated responses in retinal ganglion cells by stimulating presynaptic neurons.

Objective. Microelectronic retinal implant aims to restore functional vision with electric stimulation. Short pulses are generally known to directly activate retinal ganglion cells (RGCs) with a notion of one or two spike(s) per pulse. In the present work, we systematically explore network-mediated responses that arise from various short pulses in both normal and degenerate retinas.Approach. Cell-attached patch clamping was used to record spiking responses of RGCs in wild-type (C57BL/6J) and retinal degeneration (rd10) mice. Alpha RGCs of the mouse retinas were targeted by their large soma sizes and classified by their responses to spot flashes. Then, RGCs were electrically stimulated by various conditions such as duration (100-460µs), count (1-10), amplitude (100-400µA), and repeating frequency (10-40 Hz) of short pulses. Also, their responses were compared with each own response to a single 4 ms long pulse which is known to evoke strong indirect responses.Main results. Short pulses evoked strong network-mediated responses not only in both ON and OFF types of RGCs in the healthy retinas but also in RGCs of the severely degenerate retina. However, the spike timing consistency across repeats not decreased significantly in therd10 RGCs compared to the healthy ON and OFF RGCs. Network-mediated responses of ON RGCs were highly dependent on the current amplitude of stimuli but much less on the pulse count and the repetition frequency. In contrast, responses of OFF RGCs were more influenced by the number of stimuli than the current amplitude.Significance. Our results demonstrate that short pulses also elicit indirect responses by activating presynaptic neurons. In the case of the commercial retinal prostheses using repeating short pulses, there is a possibility that the performance of clinical devices is highly related to the preserved retinal circuits. Therefore, examination of surviving retinal neurons in patients would be necessary to improve the efficacy of retinal prostheses.

Ultrasound stimulation for non-invasive visual prostheses.

Globally, it is estimated there are more than 2.2 billion visually impaired people. Visual diseases such as retinitis pigmentosa, age-related macular degeneration, glaucoma, and optic neuritis can cause irreversible profound vision loss. Many groups have investigated different approaches such as microelectronic prostheses, optogenetics, stem cell therapy, and gene therapy to restore vision. However, these methods have some limitations such as invasive implantation surgery and unknown long-term risk of genetic manipulation. In addition to the safety of ultrasound as a medical imaging modality, ultrasound stimulation can be a viable non-invasive alternative approach for the sight restoration because of its ability to non-invasively control neuronal activities. Indeed, recent studies have demonstrated ultrasound stimulation can successfully modulate retinal/brain neuronal activities without causing any damage to the nerve cells. Superior penetration depth and high spatial resolution of focused ultrasound can open a new avenue in neuromodulation researches. This review summarizes the latest research results about neural responses to ultrasound stimulation. Also, this work provides an overview of technical viewpoints in the future design of a miniaturized ultrasound transducer for a non-invasive acoustic visual prosthesis for non-surgical and painless restoration of vision.

Artificial Visual Information Produced by Retinal Prostheses.

Numerous retinal prosthetic systems have demonstrated somewhat useful vision can be restored to individuals who had lost their sight due to outer retinal degenerative diseases. Earlier prosthetic studies have mostly focused on the confinement of electrical stimulation for improved spatial resolution and/or the biased stimulation of specific retinal ganglion cell (RGC) types for selective activation of retinal ON/OFF pathway for enhanced visual percepts. To better replicate normal vision, it would be also crucial to consider information transmission by spiking activities arising in the RGC population since an incredible amount of visual information is transferred from the eye to the brain. In previous studies, however, it has not been well explored how much artificial visual information is created in response to electrical stimuli delivered by microelectrodes. In the present work, we discuss the importance of the neural information for high-quality artificial vision. First, we summarize the previous literatures which have computed information transmission rates from spiking activities of RGCs in response to visual stimuli. Second, we exemplify a couple of studies which computed the neural information from electrically evoked responses. Third, we briefly introduce how information rates can be computed in the representative two ways - direct method and reconstruction method. Fourth, we introduce in silico approaches modeling artificial retinal neural networks to explore the relationship between amount of information and the spiking patterns. Lastly, we conclude our review with clinical implications to emphasize the necessity of considering visual information transmission for further improvement of retinal prosthetics.

Fabrication of High-Density Out-of-Plane Microneedle Arrays with Various Heights and Diverse Cross-Sectional Shapes.

Out-of-plane microneedle structures are widely used in various applications such as transcutaneous drug delivery and neural signal recording for brain machine interface. This work presents a novel but simple method to fabricate high-density silicon (Si) microneedle arrays with various heights and diverse cross-sectional shapes depending on photomask pattern designs. The proposed fabrication method is composed of a single photolithography and two subsequent deep reactive ion etching (DRIE) steps. First, a photoresist layer was patterned on a Si substrate to define areas to be etched, which will eventually determine the final location and shape of each individual microneedle. Then, the 1st DRIE step created deep trenches with a highly anisotropic etching of the Si substrate. Subsequently, the photoresist was removed for more isotropic etching; the 2nd DRIE isolated and sharpened microneedles from the predefined trench structures. Depending on diverse photomask designs, the 2nd DRIE formed arrays of microneedles that have various height distributions, as well as diverse cross-sectional shapes across the substrate. With these simple steps, high-aspect ratio microneedles were created in the high density of up to 625 microneedles mm-2 on a Si wafer. Insertion tests showed a small force as low as ~ 172 µN/microneedle is required for microneedle arrays to penetrate the dura mater of a mouse brain. To demonstrate a feasibility of drug delivery application, we also implemented silk microneedle arrays using molding processes. The fabrication method of the present study is expected to be broadly applicable to create microneedle structures for drug delivery, neuroprosthetic devices, and so on.

Spiking Characteristics of Network-Mediated Responses Arising in Direction-Selective Ganglion Cells of Rabbit and Mouse Retinas to Electric Stimulation for Retinal Prostheses.

To restore the sight of individuals blinded by outer retinal degeneration, numerous retinal prostheses have been developed. However, the performance of those implants is still hampered by some factors including the lack of comprehensive understanding of the electrically-evoked responses arising in various retinal ganglion cell (RGC) types. In this study, we characterized the electrically-evoked network-mediated responses (hereafter referred to as electric responses) of ON-OFF direction-selective (DS) RGCs in rabbit and mouse retinas for the first time. Interestingly, both species in common demonstrated strong negative correlations between spike counts of electric responses and direction selective indices (DSIs), suggesting electric stimulation activates inhibitory presynaptic neurons that suppress null direction responses for high direction tuning in their light responses. The DS cells of the two species showed several differences including different numbers of bursts. Also, spiking patterns were more heterogeneous across DS RGCs of rabbits than those of mice. The electric response magnitudes of rabbit DS cells showed positive and negative correlations with ON and OFF light response magnitudes to preferred direction motion, respectively. But the mouse DS cells showed positive correlations in both comparisons. Our Fano Factor (FF) and spike time tiling coefficient (STTC) analyses revealed that spiking consistencies across repeats were reduced in late electric responses in both species. Moreover, the response consistencies of DS RGCs were lower than those of non-DS RGCs. Our results indicate the species-dependent retinal circuits may result in different electric response features and therefore suggest a proper animal model may be crucial in prosthetic researches.

A 4.49nW/Pixel Light-to-Stimulus Duration Converter-Based Retinal Prosthesis Chip.

This paper presents a 288-pixel retinal prosthesis (RP) chip implemented in a 0.18 µm CMOS process. The proposed light-to-stimulus duration converter (LSDC) and biphasic stimulator generate a wide range of retinal stimuli proportional to the incident light intensity at a low supply voltage of 1V. The implemented chip shows 25.5 dB dynamic stimulation range and the state-of-the art low power consumption of 4.49 nW/pixel. Ex-vivo experiments were performed with a mouse retina and patch-clamp recording. The electrical artifact recorded by the patch electrode demonstrates that the proposed chip can generate electrical stimuli that have different pulse durations depending on the light intensity. Correspondingly, the spike counts in a retinal ganglion cell (RGC) were successfully modulated by the brightness of the light stimuli.

Preclinical study to improve microbubble-mediated drug delivery in cancer using an ultrasonic probe with an interchangeable acoustic lens.

Focused ultrasound with microbubbles (FUS-MBs) has shown that it can lead to an efficient drug delivery system (DDS) involving the oscillation and destruction of the MB but is limited in drug delivery due to its narrow pressure field. However, unfocused ultrasound with MBs (UUS-MBs) and an interchangeable acoustic lens can tune and enhance the pressure field for MB destruction to overcome the disadvantages of FUS-MB DDSs. We designed a lens suitable for an ultrasound-phased array probe and studied the optimal treatment conditions for MB destruction in vitro through an optical imaging setup. The DDS effects were evaluated in a rat hepatoma model using doxorubicin (DOX) treatment. A concave lens with a radius of curvature of 2.6 mm and a thickness of 4 mm was selected and fabricated. UUS-MBs with the acoustic lens at 60 Vpp for 32 cycles and a PRF of 1 kHz could induce MB destruction, promoting the DDS even under fluidic conditions. In the animal experiment, the UUS-MBs in the acoustic lens treatment group had a higher concentration of DOX in the tumor than the control group. Our system suggests uses an acoustic lens to increase DDS effectiveness by providing sufficient ultrasound irradiation to the MBs.

Electric Stimulation Elicits Heterogeneous Responses in ON but Not OFF Retinal Ganglion Cells to Transmit Rich Neural Information.

Retinal implants electrically stimulate surviving retinal neurons to restore vision in people blinded by outer retinal degeneration. Although the healthy retina is known to transmit a vast amount of visual information to the brain, it has not been studied whether prosthetic vision contains a similar amount of information. Here, we assessed the neural information transmitted by population responses arising in brisk transient (BT) and brisk sustained (BS) subtypes of ON and OFF retinal ganglion cells (RGCs) in the rabbit retina. To correlate the response heterogeneity and the information transmission, we first quantified the cell-to-cell heterogeneity by calculating the spike time tiling coefficient (STTC) across spiking patterns of RGCs in each type. Then, we computed the neural information encoded by the RGC population in a given type. In responses to light stimulation, spiking activities were more heterogeneous in OFF than ON RGCs (STTCAVG = 0.36, 0.45, 0.77 and 0.55 for OFF BT, OFF BS, ON BT, and ON BS, respectively). Interestingly, however, in responses to electric stimulation, both BT and BS subtypes of OFF RGCs showed remarkably homogeneous spiking patterns across cells (STTCAVG = 0.93 and 0.82 for BT and BS, respectively), whereas the two subtypes of ON RGCs showed slightly increased populational heterogeneity compared to light-evoked responses (STTCAVG = 0.71 and 0.63 for BT and BS, respectively). Consequently, the neural information encoded by the electrically-evoked responses of a population of 15 RGCs was substantially lower in the OFF than the ON pathway: OFF BT and BS cells transmit only ~23% and ~53% of the neural information transmitted by their ON counterparts. Together with previously-reported natural spiking activities in ON RGCs, the higher neural information may make ON responses more recognizable, eliciting the biased percepts of bright phosphenes.

Editorial for the Special Issue on Micro/Nanofabrication for Retinal Implants.

The retinal prosthetic community has witnessed tremendous technological advances during the last two decades since the emergence of pioneering work [...].

Versatile Low-Cost Volumetric 3D Ultrasound Imaging Using Gimbal-Assisted Distance Sensors and an Inertial Measurement Unit.

This study aims at creating low-cost, three-dimensional (3D), freehand ultrasound image reconstructions from commercial two-dimensional (2D) probes. The low-cost system that can be attached to a commercial 2D ultrasound probe consists of commercial ultrasonic distance sensors, a gimbal, and an inertial measurement unit (IMU). To calibrate irregular movements of the probe during scanning, relative position data were collected from the ultrasonic sensors that were attached to a gimbal. The directional information was provided from the IMU. All the data and 2D ultrasound images were combined using a personal computer to reconstruct 3D ultrasound image. The relative position error of the proposed system was less than 0.5%. The overall shape of the cystic mass in the breast phantom was similar to those from 2D and sections of 3D ultrasound images. Additionally, the pressure and deformations of lesions could be obtained and compensated by contacting the probe to the surface of the soft tissue using the acquired position data. The proposed method did not require any initial marks or receivers for the reconstruction of a 3D ultrasound image using a 2D ultrasound probe. Even though our system is less than $500, a valuable volumetric ultrasound image could be provided to the users.

Retinal Degeneration Reduces Consistency of Network-Mediated Responses Arising in Ganglion Cells to Electric Stimulation.

Retinal prostheses use periodic repetition of electrical stimuli to form artificial vision. To enhance the reliability of evoked visual percepts, repeating stimuli need to evoke consistent spiking activity in individual retinal ganglion cells (RGCs). However, it is not well known whether outer retinal degeneration alters the consistency of RGC responses. Hence, here we systematically investigated the trial-to-trial variability in network-mediated responses as a function of the degeneration level. We patch-clamp recorded spikes in ON and OFF types of alpha RGCs from r d10 mice at four different postnatal days (P15, P19, P31, and P60), representing distinct stages of degeneration. To assess the consistency of responses, we analyzed variances in spike count and timing across repeats of the same stimulus delivered multiple times. We found the trial-to-trial variability of network-mediated responses increased considerably as the disease progressed. Compared to responses taken before degeneration onset, those of degenerate retinas showed up to ~70% higher variability (Fano Factor) in spike counts (p < 0.001) and ~95% lower correlation level in spike timing (p < 0.001). These results indicate consistency weakens significantly in electrically-evoked network-mediated responses and therefore raise concerns about the ability of microelectronic retinal implants to elicit consistent visual percepts at advanced stages of retinal degeneration.