Optimizing Electrical Stimulation Parameters to Enhance Visual Cortex Activation in Retina Degeneration Rats

doi:10.21203/rs.3.rs-4724312/v1

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Optimizing Electrical Stimulation Parameters to Enhance Visual Cortex Activation in Retina Degeneration Rats

https://doi.org/10.21203/rs.3.rs-4724312/v1

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In patients with degenerative diseases of the retina, such as retinitis pigmentosa and age-related macular degeneration, retinal prostheses offer a possible method of restoring partial vision. Epiretinal prosthesis remain the need for optimal stimulation parameters for better efficiency and power saving while showing encouraging preliminary clinical efficacy. In this study, we investigate the effects of retinal electrical stimulation parameters, including phase duration, frequency, and interphase interval, on the visual cortical electrically evoked potentials (EEPs) in both healthy (LE) and retinal degenerated (F1) rats. Our in vivoexperiments on both LE and F1 rats suggest that shorter phase durations (500 μs) activate the primary visual cortex (V1) at a lower charge threshold and result in a larger activated area. Our results also show that response to repetitive stimulation decreases profoundly at high frequencies (10 and 20 Hz) compared to low frequency (1 Hz). Additionally, we observe that the inclusion of an interphase interval enhances the level of cortical response but does not contribute to an increase in the activation ratio. The present work suggests epiretinal stimulation using shorter phase durations without interphase intervals as the optimal strategy to safely activate the target region in V1 in both LE and F1 rats.

Health sciences/Diseases/Eye diseases/Vision disorders

Health sciences/Diseases/Neurological disorders/Neurodegeneration

Biological sciences/Physiology/Neurophysiology

Retinal degeneration

retinal stimulation

stimulation parameters

cortical response

rat

Patients who have been blinded by retinal degenerative diseases, such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP), can regain functional vision with the help of retinal implants ^1–3. The principle stems from early research that discovered the survival of the majority of cells in the inner nuclear cell layer and the residual ganglion cells in the retina of patients with retinal degeneration such as RP. This finding illustrates the potential for vision restoration, bypassing damaged photoreceptors, by directly stimulating the surviving inner retinal neurons ^1–5. Clinical studies on retinal implants suggest that stimulating undamaged neurons in the retina could restore partial vision, even years after the onset of complete blindness in RP patients ^6,7. The previously marketed epiretinal prostheses, Argus II (Second Sight Medical Products Inc.) and IRIS II (Pixium Vision SA), as well as the subretinal prosthesis Alpha AMS (Retina Implant AG), have been discontinued ². Several technologically advanced retinal prostheses are currently being evaluated for clinical use. These include the NR600 epiretinal prosthesis by Nano Retina, Rainbow Medical Group, and the PRIMA subretinal prosthesis by Pixium Vision SA ^8–10. Additionally, there are ongoing clinical trials for two suprachoroidal prostheses in Australia by Bionic Vision Technologies ¹¹ and in Japan by Osaka University ¹², indicating a possible turning point in the development of visual prostheses. Patients with retinal degeneration, aided by retinal prostheses, have been shown to perceive spots of light, recognize objects and letters, and even improve their orientation and mobility. Furthermore, long-term implantation and electrical stimulation of these prostheses have been demonstrated to be relatively safe ^2,13–18.

Despite showing promising preliminary clinical results, retinal prostheses are still limited by several factors that constrain their overall performance, including rudimentary resolution for critical vision ². These limitations are influenced by various factors, such as the number and size of electrodes in a multielectrode array (MEA), the distance between the electrode surface and the retina, and the type of stimulation used ^2,19. A clinical study focusing on retinal prostheses revealed that, in a sample of 30 patients equipped with these prostheses, only 55% of the electrodes could elicit visual perceptions when utilizing a stimulus fell below the charge density limit for the electrode ^18,20. Therefore, there remains a clinical need to optimize the stimulation strategy of retinal implants to enhance efficiency and improve the performance evoked in the visual cortex. Several preclinical studies indicate that neural response amplitude and power efficiency can be influenced by various stimulus parameters, such as pulse duration and waveform shape ^19,21–26. However, only a few previous studies have examined the in vivo electrophysiological response to epiretinal retinal electrical stimulation at the cortical level ^25,27–30. Cortical activity maps demonstrate retinotopy in normal rats when epiretinal stimulation is applied to specific quadrants of the retina, but this phenomenon is not observed in rats with retinal degeneration ²⁹. An in vivo study reported that the retinotopy observed in cortical activity maps of rats which is likely due to a combination of factors, including the degeneration itself and the corresponding increase in stimulus thresholds ²⁹. The pathophysiology of retinal degeneration suggests that when assessing the efficiency of retinal stimulation, comparing the effect of stimulation parameters in both normal and retinal degeneration models would be more beneficial. Moreover, it has been reported that local field potential (LFP) can serve as a reliable signal for investigating the spread of cortical activity in response to retinal stimulation ³¹

In this study, the optimization of retinal stimulation efficiency is investigated by evaluating the LFPs in the visual cortex of both healthy Long Evans (LE) rats and retinal degenerated S334ter-line-3 (F1) rats in vivo, which is achieved through electrical stimulation of the retina using different parameters such as pulse duration, stimulus frequency, and interphase-interval.

2.1 Animals

Eight adult LE rats and five adult F1 rats were used in this study as wild-type and retinal degeneration models, respectively. All the rats in the two groups used in the experiments were between the ages of 7 and 10 weeks and weighed 300–500 g. The LE rats were obtained from the Laboratory Animal Services Centre of the Chinese University of Hong Kong and the F1 rats were acquired from School of Medicine of University of California, San Francisco. All animals were housed in the Laboratory Animal Research Unit at City University of Hong Kong. The animals were kept on a 12-h light/dark cycle, and food and water were available to them ad libitum. Upon completion of experiments, animals were humanely euthanized by overdose of anaesthetic (Pentobarbitone, 150 mg/kg, i.p.). All experimental procedures were approved by the Animal Subjects Ethics Sub-Committees of City University of Hong Kong (3A-3-201202) and the Health Department of the Hong Kong Special Administrative Region (11–17 in DH/HA&P/8/2/5 Pt.1). Experiments in this study complied with the ARRIVE guidelines. All experiments were performed in accordance with relevant guidelines and regulations.

2.2 Epiretinal Electrical Stimulation Preparation

Rats were initially anesthetized by intraperitoneal injection with a mixture of ketamine and xylazine (Ketamine: 70 mg/kg, and Xylazine: 7 mg/kg; Alfasan International B.V., Holland) and then mounted in a stereotaxic device. Throughout the surgical procedure and subsequent neural recording, anesthesia was maintained with isoflurane gas, administered with an anesthesia machine (SurgiVet, Smiths Medical PM, Inc., USA). The subject’s body temperature was monitored and maintained at approximately 37.5°C by a heating pad (Model # TP702; Gamry Industries, Inc., NY, USA). Prior to surgery, the stimulated eye of the pupil was dilated with several drops of tropicamide (Mydrin-P, Santen Pharmaceutical Co. Ltd., Osaka, Japan). During surgery and recording, the stimulated eye was periodically smeared with eye gel (Lubrithal, Dechra Veterinary Products A/S, Mekuvej, Denmark) to keep it clear and moist.

A rubber membrane, measuring 2 cm on each side and equipped with a small central hole was utilized to protrude the eyeball to facilitate the insertion of a stimulating electrode. The entire insertion procedure was operated under a microscope. A needle was employed to incise the upper sclera, thereby creating a pathway for the insertion of the bipolar concentric stimulating electrode (Pt/Ir, diameter of 75 µm, FHC Inc., Bowdoin, ME, USA). Following this, the stimulating electrode was inserted through the incision site along the established path, aided by an articulated holder (Noga, Noga Engineering Ltd., Israel) equipped with calipers. To ensure that the tip of the stimulating electrode was close enough to the epiretinal surface, a potentiostat (Reference 600, Gamry Instruments, Warminster, USA) was used to measure the electrode-retinal impedance in order to estimate the distance between the electrode and the retina surface. Similar operations of electrode implantation and impedance monitoring were introduced in previous studies ^32–34.

2.3 Electrophysiological Signal Recording

A craniotomy was performed on the occipital lobe (approximately 2.5–5.5 mm lateral to the midline and 4.5–7.5 mm posterior to the bregma). This site corresponds to the visual cortex contralateral to the stimulated eye. Following the procedure, the skull piece was carefully removed using a hand-held drill, after which the dura was also gently extracted with a needle. After craniotomy, a bone screw was implanted in the skull to serve as the ground electrode. Additionally, a 4×4 grid electrode array (Clunbury Scientific LLC., Bloomfield Hills, USA), comprising 16 electrodes each measuring 3 mm in length, with a 400 µm distance between each tip, was employed to record in vivo electrophysiological signals. This recording electrode array was inserted into the exposed primary visual cortex, approximately 800–950 µm deep, using a microdrive inserter (Micropositioner, David Kopf Instruments, Tujunga, USA) (Fig. 1(a)). Following craniotomy, the rats were given an hour to recover before being stimulated.

The response to retinal electrical stimulation, defined as electrically evoked potentials (EEPs), was recorded from the primary visual cortex (V1), contralateral to the stimulated eye (Fig. 1(a)). In this study, we employed LFPs to investigate visual cortical responses, primarily focusing on the network-mediated response. Using a data acquisition system (Micro1401, CED, UK) and an amplifier (A-M system, Inc. Model 3600, UK), the EEPs were recorded at a sampling frequency of 25 kHz with a band-passed filter of 0.3–300 Hz.

2.4 Impedance Measurement and Electrical Stimulation of the Retina

In this study, the stimulation electrode was positioned in the ventral-temporal region of the retina using an articulated holder equipped with calipers. Subsequently, the impedance between the electrode and the retinal surface was measured in real-time by a potentiostat deploying a 10-mV (r.m.s.) AC sinusoid signal at 100 kHz. This impedance served as a measure for evaluating the distance between the electrode and the retinal surface. Previous studies have established the relationship between impedance and the electrode-epiretinal distance, as well as EEPs, in rats ^32–34. These studies showed that the impedance monotonically decreased as the electrode moved closer to the epiretinal surface. Moreover, when the impedance was valued between 5–8 kΩ, it correlated well with visual cortical responses, which however, showed a decrease when the impedance value exceeded 8 kΩ. In all experiments conducted in this study, the electrode was manipulated to alter the distance between it and the epiretinal surface until the impedance value consistently measured around 8 kΩ. Prior experiments have determined this to be the optimal impedance value to ensure safe and efficient stimulation while preserving the integrity of the implant. These studies also suggest that an impedance value of 8 kΩ signifies a distance of approximately 370 µm between the electrode and epiretinal surface ³⁴.

Charge-balanced, cathodic first, biphasic current stimulations, ranging from 10 to 200 µA in increments of 10 µA (Fig. 1(b), each current repeated 10 times), were applied to the epiretinal surface using a stimulus generator (STG 2004, Multichannel Systems MCS GmBH, Germany). The reference electrode was inserted into the skin adjacent to the nose. Two stimulus patterns were used: one without an interphase interval (IPI) (Fig. 1(b)) and one with an IPI (Fig. 1(c)). The pattern without an IPI utilized four frequencies (1, 5, 10 and 20 Hz) and four phase durations (200, 500, 1000 and 1500 µs). In contrast, the pattern with an IPI used only a frequency of 5 Hz and a phase duration of 1000 µs. The IPI involved four different lengths: 0 (i.e., without an IPI), 500, 1000 and 1500 µs. Each parameter set underwent two retinal stimulation trials. Rats were allowed a 5-min recovery interval between trials to avoid retinal damage.

2.5 Characterization of Electrical Evoked Potentials of Visual Cortical Neurons

The amplitude from the first negative peak to baseline was used as the response level to analyze the effects of varying stimulation parameters and stimulus patterns on cortical neural responses (Fig. 1(d)). Specifically, the first negative peak was defined as the minimum point in the timeframe from 10 ms to 50 ms post-stimulation. The response level was represented by a fitted sigmoid curve plotted as a function of varying current amplitude (ranging from 10 to 200 µA in increments of 10 µA). The activation threshold was defined as the current that evoked 50% (P50) of the saturated response (Fig. 1(e)).

To evaluate the effectiveness of various electrical retinal stimulation parameters, the responses evoked by different parameter sets were compared. Additionally, a comparison of response levels between the LE and F1 groups was performed to further examine the impact of stimulation parameters on rats with retinal degeneration. To examine the spatially activated distribution, the activated area in the V1 was visualized using an interpolated heat map (Fig. 2(a)) and the activation ratio was calculated by converting the heat map into a binary map (Fig. 2(b)). Figure 2 represents heat maps and binary maps illustrating the response of a single rat to a range of stimulus currents (50, 100, 150, and 200 µA). The arrangement of the 16 recording electrodes was represented by the 4×4 array shown in the map. Responses were initially averaged across the ten trials, and subsequently, each rat’s response was normalized to the maximum response (at 200 µA). As indicated in Fig. 2(a), regions on the heat map closer to the dark red range exhibited a stronger response as compared to those nearer to the dark blue range. Figure 2(b) illustrated the binary map where the region with a response level surpassing half of the maximal value is identified as the activated area, while the rest is categorized as the inactivated area. To evaluate the effectiveness of retinal stimulation with different stimulation parameters, the activation ratio was calculated. This ratio was defined as the proportion of the activated area to the entire region. For the statistical analysis of the activation ratio, self-normalization was implemented to mitigate variations among different animals.

2.6 Data Processing and Analysis

The Spike2 software (CED, UK) was employed to acquire the signal, and subsequently, the data was processed and analyzed offline using MATLAB. Comparison of the response levels across all 16 channels was conducted and the channel exhibiting the largest response was chosen for further analysis. The goodness of fit of the sigmoid function was calculated to exclude data with a fit lower than 60%, indicating no response. Unpaired two-tailed Student’s t-test and ANOVA were employed for statistical analysis. One F1 rat did not yield a response signal due to the malfunction of the recording electrode array during the experiment. As a result, the analysis comprised of a total of eight LE rats (16 trials, 2 repeated trials for one experiment) and four F1 rats (8 trials).

Sections 3.1 to 3.3 focused on the effect of phase duration and frequency without IPI on threshold (Section 3.1), activation ratio (Section 3.2), and response level (Section 3.3). On the other hand, sections 3.4 and 3.5 were dedicated to investigating the addition of IPI, analyzing its effect on the visual cortical response in terms of response level (Section 3.4) and activation ratio (Section 3.5). All subsections included a comparison between the LE and F1 groups.

3.1 Effect of Stimulation Phase Duration and Frequency on Threshold Current and Charge

The effect of phase duration on the current and charge threshold was examined in this study using the stimulation pattern devoid of an interphase interval (Fig. 3). The charge threshold was obtained by multiplying the current stimulus threshold by the phase duration. This finding revealed that the F1 group exhibited a higher activation threshold compared to the LE group, in agreement with previous research ^33,35,36. The thresholds of the LE and the F1 groups were also compared at various stimulation frequencies, such as 1, 5, and 10 Hz. The threshold-duration curve was shown in Fig. 3. Specifically, Fig. 3(a)-(c) demonstrated that the current threshold typically decreased with an increase in the stimulation phase duration. In contrast to the current threshold, an increase in the phase duration resulted in an upward trend in the corresponding charge threshold (Fig. 3(d)-(f)).

At a stimulation frequency of 1 Hz, there was a significant difference observed between the LE and F1 groups in terms of both charge threshold and current threshold (Fig. 3(a) and 3(d)), which was not evident at 5 Hz (Fig. 3(b) and 3(e)) or 10 Hz (Fig. 3(c) and 3(f)). Fig. 3(a) and 3(d) showed a noticeable difference in the current threshold and charge threshold between the LE and F1 group under 1 Hz stimulation with 1000 and 1500 µs phase duration (unpaired t-test, ∗p < 0.05). A more significant difference was observed in both the current threshold and charge threshold between the LE (76.94±11.33 µA and 38.47±5.67 nC, 16 trials, 8 rats) and F1 group (161.56±17.00 µA and 80.78±8.50 nC, 8 trials, 4 rats) (unpaired t-test, ∗∗p < 0.01) at 1 Hz stimulation with 500 µs phase duration (Fig. 3(a) and 3(d)). At 5 Hz (Fig. 3(b) and 3(e)), only the threshold of 1000 µs phase duration had apparent difference between the LE (94.85±8.19 µA and 94.85±8.19 nC, 16 trials, 8 rats) and F1 group (132.92±14.35 µA and 132.92±14.35 nC, 8 trials, 4 rats) (unpaired t-test, ∗p < 0.05). As the stimulation frequency increased to 10 Hz (Fig. 3(c) and 3(f)), there was no significant difference between the LE and the F1 groups. These results suggested that the degeneration of the retina may influence the sensitivity of retinal cells to low-frequency stimulation.

3.2 Effect of Phase Duration and Frequency on Spatial Activation in the Primary Visual Cortex

The activation ratio in V1 was analyzed in this section to explore the effect of the phase duration (500, 1000, and 1500 µs) and the frequency (1, 5, and 10 Hz) on the activation ratio of visual cortical response. The binary map (Fig. 4(a)) displayed the activated and inactivated area and showed the activation ratio. The activation ratio used for statistical analysis was normalized to a value of 1.0 for the stimulation with 500 µs phase duration within each animal to reduce the variance among different animals.

Fig. 4(a) showed examples of binary maps with different stimulation phase durations at 200 μA. Fig. 4(b)-(e) represented bar charts depicting the activation ratio evoked by threshold current and suprathreshold (i.e., 200 µA) for the LE and F1 groups, respectively. Although the activation ratio generally declined with pulse duration increasing at threshold current in the LE group (Fig. 4(b)), there was no statistical difference at 1 Hz stimulation in response to the stimulation at threshold current (ANOVA, ∗p < 0.05, 16 trials, 8 rats). To ensure complete activation of the cortical neurons, we further computed the activation ratio of these neurons in response to suprathreshold (i.e., 200 µA) stimulation (Fig. 4(c)). The ANOVA analysis showed that, at 1 Hz stimulation, the activation ratio decreased significantly from 1 for a 500 µs phase duration to 0.74±0.09 for a 1500 µs phase duration in LE group (ANOVA, ∗p < 0.05, 16 trials, 8 rats). Significant differences were also observed between the activation ratios elicited by 500 µs phase duration and 1500 µs phase duration (0.52±0.10 for 5 Hz, 0.36±0.15 for 10 Hz) (ANOVA, ∗p < 0.05, 16 trials, 8 rats).

As for the F1 group, no significant difference was observed in the activation ratio under threshold current stimulation (Fig. 4(d)). When the stimulation current was increased to the suprathreshold level of 200 µA, the cortical activation ratio in the F1 group exhibited trends similar to those in the LE group. Specifically, the activation ratio decreased as the phase duration increased (Fig. 4(e)).

3.3 The Cortical Response Level Declined with Increasing Epiretinal Stimulation Frequency Under Equal Charge Injection

The effect of stimulation frequency on cortical response level was investigated in this section. Separate plots of frequency versus charge contour figures (Fig. 5(a) and 5(b)) were generated to examine the overall trend of the impact of stimulation frequency on the response levels of EEPs in the LE and F1 groups, respectively. To determine the charge, the current was multiplied by the duration of a single phase. At 200 μA stimulation, four distinct charges (40, 100, 200 and 300 nC) were computed for varying phase durations. Under the same charge injection scale, it was observed that the response level was suppressed with stimulation frequency increasing, as shown in Fig. 5(c)-(f).

The cortical response level induced by electrical stimulation exhibited a decreasing trend with increasing stimulation frequency, regardless of the charge amount, as evidenced by the results of each of the four phase durations. With a charge injection of 40 nC, as the stimulation frequency increased from 1 to 20 Hz (Fig. 5(c)), the response level decreased from 195.39±74.56 μV and 71.87±55.39 μV to 49.41±14.38 μV and 16.21±2.90 μV for the LE (16 trials, 8 rats) and F1 (8 trials, 4 rats) groups, respectively. With a charge injection of 300 nC (Fig. 5(f)), the response level was 599.37±111.50 μV for the LE (16 trials, 8 rats) group and 426.91±184.47 μV for the F1 (8 trials, 4 rats) group at 1 Hz stimulation, and decreased to 63.28±17.25 μV for the LE group and 102.29±44.41 μV for the F1 group at 20 Hz stimulation. This finding was consistent with the results of previous studies that investigated the optic nerves of rabbits ^37,38. Although the unpaired t-test did not indicate a significant difference between the LE and F1 groups, there was a noticeable distinction between the two groups. Specifically, the LE group exhibited a more pronounced difference in response level than the F1 group for the four typical injecting charges (40, 100, 200 and 300 nC) at 1 Hz stimulation (Fig. 5(g)), while responses of both groups were similar at high stimulation frequencies (10 and 20 Hz) (Fig. 5(i)-(j)). Notably, with a charge injection of 300 nC, the response level of the LE group even dropped below that of the F1 group at 10 and 20 Hz (Fig. 5(i)-(j)).

3.4 Effect of Interphase Interval on Evoked Response Amplitude in Retinal Electrical Stimulation

The effect of interphase intervals of electrical retinal stimulation on the cortical response level was investigated in this section. Our results showed that thresholds in response to the stimulation with different IPIs in the F1 group were higher than that in the LE group. Yet no significant difference was found between the two groups (Fig. 6(a)). As we aimed to explore the impact of inter-phase interval on the cortical response, the response level and activation ratio evoked by stimuli with various inter-phase intervals were compared at the same stimulation current amplitude in F1 and LE groups, respectively. To examine the effect of the interphase interval, Fig. 6(b)-(c) showed the interpolated contour plots for EEPs at 5 Hz stimulation with 1000 µs phase duration. The IPI values used in this investigation are 0, 500, 1000 and 1500 μs. Our results showed that a longer IPI can elicit a higher response level with the same stimulation current in LE group. In the F1 group, the introduction of IPI also increased the response level, while the response level strengthened with the increment of IPI only observed from 500 to 1000 μs. The impact of IPI peaked at 1000 μs and became weaker at 1500 μs. Moreover, the F1 group necessitated a longer IPI and a higher current level to produce a neural signal of the same magnitude as the LE group. Our results demonstrated that adding the IPI during electrical stimulation could elicit a stronger response at the cortical level.

3.5 Effect of Interphase Interval on Spatial Activation

To examine the effect of stimulation IPI on cortical EEPs, we analyzed the spatial responses by determining the activation ratio of cortical response elicited by this particular form of stimulation. Fig. 7(a) displayed a representation of the spatial activation of EEPs within the visual cortex in response to retinal stimulation at 200 µA, with different IPI durations (0, 500, 1000 and 1500 μs) normalized to the maximum response, highlighting the activation pattern. The top row showed the heat map and the bottom row showed the corresponding binary map plotted based on 50% of the maximum responses. As the IPI duration increased, the activation ratio decreased correspondingly. Additionally, contour figures were generated to visualize the activation ratio of EEPs versus the IPI and the stimulation current for both the LE and F1 groups in Fig. 7(b)-(c). Fig. 7(b) illustrated that, under identical stimulation current, the shorter IPI evoked a greater response in the LE group. A comparable trend was observed in the F1 group, as depicted in Fig. 7(c).

Quantitative analysis for spatial characteristics was performed using the responses elicited by threshold current and 200 μA in the LE (Fig. 7(d) and 7(e)) and F1 groups (Fig. 7(f) and 7(g)). The activation ratio used for statistical analysis was normalized to a value of 1.0 for the stimulation without IPI within each animal to reduce the variations among different animals. The response to current threshold showed that the addition of IPI diminished the activation ratio in both LE (Fig. 7 (d)) and F1 groups (Fig. 7 (f)). However, zero registrations at IPI of 1000 μs and 15000 μs indicate weak responses in certain experimental sets (Fig. 7 (f)). Considering that the response to 200 μA was maximum and saturated, further analysis of spatial activation was performed at 200 μA to better illustrate the results. The results showed an overall decreasing trend in activation ratio for both LE and F1 groups as the IPI increased (Fig. 7(d) and 7(e)). Specifically, in LE group (16 trials, 8 rats; Fig. 7(d)), the activation ratio declined significantly from 1 to 0.80±0.05 (for 500 μs IPI, ANOVA, ∗∗p < 0.01), 0.71±0.06 (for 1000 μs IPI, ANOVA, ∗∗p < 0.01), and 0.72±0.09 (for 1500 μs IPI, ANOVA, ∗∗p < 0.01). As for F1 group, Fig. 7(g) showed that at 200 μA stimulation, the activation ratio evoked by retinal stimulation without IPI was significantly higher than that elicited by stimulation with 1000 μs and 1500 μs. These results suggest that adding IPI does not enhance the effectiveness of retinal stimulation on cortical spatial response in both the LE and F1 groups.

This study demonstrates that utilizing electrical stimulation on epi-retina with a short phase duration (500 µs) can lower the charge threshold required to activate the visual cortical neurons and achieve a broader spatial activation area in both LE and F1 rats. Our experiments reveal no contribution of IPI to spatial activation and threshold, though the response level increases with an increment of IPI. All results were obtained from in vivo experiments with rats exhibiting retinal degeneration, with almost entirely lost photoreceptors, and rats with normal eyesight. Our findings suggest a potential epiretinal stimulation strategy that can efficiently evoke a targeted cortical response without causing tissue injury.

Visual implants hold promise for partially restoring vision in patients with retinal degeneration ^{2,4,9,10,39–44}. However, these devices still face significant limitations, including the low resolution of artificial vision ^44–47. Therefore, it is crucial to improve stimulation strategies to enhance the effectiveness of stimulation. This improvement can have far-reaching benefits, not only for visual prosthetics but also for other areas within the field of neuroprosthetics. Improving retinal implant performance and minimizing the risk of retinal tissue injury remains a significant challenge. Weitz et al.'s in vitro research demonstrated that pulses of 25 ms and longer could evoke responses in RD retinas, even in cases where there was a complete loss of photoreceptors. Furthermore, their study suggested that pulses of 25 ms and longer could selectively activate inner neurons by lowering their activation threshold ³⁵. Similarly, this study aims to optimize the stimulation parameters to reduce thresholds, making it more feasible to stimulate neurons without causing harm to the neural tissue and electrodes.

In addition, employing an optimal stimulation strategy with a lower charge threshold can also reduce power consumption for the implants, making it a practical consideration. However, there have been limited in vivo studies conducted on both LE and retinal degenerate F1 animals to examine the effects of electrical stimulation pulse duration, frequency, and interphase interval on the V1 cortical neural response, which is the primary focus of this study. Our previous work examined the optimal distance between the stimulating electrode and the retinal surface to ensure safe and efficient stimulation based on the optimal electrode-retina impedance ³⁴. Building upon this, the present study further investigated the optimal stimulation parameters that can effectively evoke visual cortical responses from the perspective of threshold, response level, and spatial characteristics. The findings indicated that optimizing the stimulation parameters, including phase duration, stimulus frequency, and interphase interval, can reduce the threshold and generated more extended cortical activation in both LE and F1 rats. These results suggested a promising retinal stimulation strategy for achieving efficient cortical activation.

Regarding the threshold required to evoke 50% of maximum response, our results indicated that the current threshold decreased with an increase in phase duration, as demonstrated in Fig. 3(a)-(c). Conversely, the charge threshold increased with phase duration as seen in Fig. 3(d)-(f). These findings aligned with prior in vivo studies of intraorbital ON optic nerve electrical stimulation in rabbits ^37,38 as well as in vitro experiments performed in rodents’ retinas ⁴⁸. Utilizing shorter phase duration stimuli could minimize the total injecting charge required to activate retinal cells, thus reducing potential damage to neural tissue and providing a possible strategy to lower the power consumption of retinal implants. However, given the short phase duration, a relatively higher current amplitude is necessary to reach the activation threshold. This raised the need for high-current delivery electronic devices, which might be hard to design ⁴⁹.

Our aggregate results showed the LE group exhibited lower thresholds compared to the F1 group, a finding that aligns with conclusions from previous in vitro studies on isolated rodents’ retinas ^35,50,51. Cho et al. found that rd10 retinas with low spontaneous activities required an increased charge to evoke responses, while the thresholds of rd10 retinas and wild-type mice with high spontaneous activities were similar ⁵². In the present study, our results showed that the threshold difference between the two groups decreased as the stimulation frequency increased from 1 to 10 Hz, and the thresholds were nearly equal at 10 Hz (Fig. 3). Previous research suggested that low-frequency stimulation of sinusoidal waveform activates bipolar cells and/or photoreceptors ⁵³. The present study showed that at low frequency stimulation (1 Hz) of biphasic waveform, healthy group exhibited a larger response level compared to that of the degenerated group, while at high frequency stimulation (20 Hz), the difference of response level between the two groups demolished. Our finding suggested that similar retinal neurons were activated at high frequency stimulation while the low frequency stimulus activated different types of retinal neurons, using biphasic waveforms. In contrast, an earlier in vitro study reported that surviving RGCs during retinal degeneration progression functioned similarly to those in the healthy group, indicating a preferential activation of RGCs at high frequencies when the charge-balanced biphasic rectangular current stimulation was applied ⁵⁴. The comparable threshold for healthy and retinal degeneration mice was also reported by in vitro works on voltage stimulation ⁵⁵ and current stimulation at 10 Hz ⁵² and 5–10 Hz ⁵⁶. The differences might be partially attributable to retinal remodeling, though the potential changes in the intrinsic physiological characteristics of the retinal ganglion cells remained unclear. These results implied that retinal cell sensitivity to low-frequency stimulation is influenced by retinal degeneration. Considering this, we suggested the higher stimulation frequency (i.e., 10 Hz) as the efficient parameter to activate the visual cortical neurons in retinal degeneration rats at lower charge injection because larger charge injection increased power consumption and necessitated electrode materials with a high charge capacity ⁴⁴.

Furthermore, we explored the impact of IPI addition on the threshold. Our results showed no noticeable influence on the threshold for both LE and F1 rats (Fig. 6(a)). This deviates from Weitz et al.’s conclusion, which was derived from experiments on isolated salamander retinas, a computational model, and human testing ²⁴. Specifically, their study uncovered an inverse relationship between threshold and interphase gap (IPG) (also known as IPI) duration. Another study showed that IPIs shorter than phase duration did not yield significant differences ⁴⁸. Given this finding, the discrepancy between our study and Weitz et al. might be attributed to the varying ratios of IPI and phase duration. The IPIs used in Weitz et al.’s work ranged from 26–400% of phase duration, while our IPIs only extended up to 1500 µs (150% of phase duration). It requires further study to examine the efficacy of longer IPI (> 150%) in visual cortical responses.

We found that the response level of EEPs in V1 monotonically declined as the stimulation frequency increased from 1 to 20 Hz in both the LE and F1 groups (Fig. 5), regardless of the stimulation charge. A similar suppressive effect of stimulation frequency increment on visual cortical response was observed in network-mediated cortical response to repeated stimulation on the mice’s corneal surface ¹⁹. Additionally, the in vitro experiments also reported the reduction of spike activity in RGC (referred to as desensitization) in response to repetitive stimulation with frequency increase ^57,58. As the response used for analysis was averaged over 10 repeated consecutive stimulations, with frequency increased, the decrease in response level possibly resulted from desensitization due to repetitive stimulation. However, our results did not uncover the underlying mechanism behind this desensitization. Therefore, it would be intriguing to explore the source of this desensitization observed in cortical responses to repetitive retinal electrical stimulation.

The introduction of the IPI between two phases of the biphasic pulse in the present study aimed to investigate its effect on the visual cortical response to retinal electrical stimulation. Results showed that inclusion of IPI led to an increase in the response level of the EEPs in both the LE and F1 groups (Fig. 6(b)-(c)). Previous research had reported similar findings regarding the rabbits’ visual cortical and salamanders’ retinal responses to retinal electrical stimulation, indicating that the inclusion of the IPI can increase the amplitude of EEP ^24,38. We noted that the LE group exhibited higher response levels with an increase in the IPI of the biphasic stimulation. In the case of the F1 group, the beneficial effect of incorporating an IPI into the cortical response reached its maximum at an IPI of1000 µs and slightly diminished at an IPI of 1500 µs.

As reported in an early study with clinical tests, thresholds gradually increased during the test ⁵⁹. Also, the increase in stimulation amplitude made the phosphenes brighter and larger ⁴⁶, thereby elevating the need for efficient spatial activation. Building upon this, an optimal stimulation parameter is needed to activate the target region at a lower stimulation level. This could increase the dynamic range of the stimulation current adjustment and reduce the risk of retinal tissue injury. In addition to modifying the electrode configuration ^54,60–62, some earlier studies have conducted experiments on isolated retina in vitro to explore the spatial response with respect to varying pulse durations and frequencies ^35,36,63. Additionally, in vivo research was conducted on cats and rabbits to examine the influence of stimulation parameters on visual cortical response. Su et al.’s group employed optical intrinsic signal imaging to evaluate the spatial activation area in the healthy area 18 of the occipital cortex (also known as secondary visual cortex) in cats, which was stimulated by varying frequencies, intensities, and pulse durations ⁶⁴. In Sun et al.’s study, they collected electrophysiological signals from healthy rabbits V1 and investigated the correlation between spatial activation of cortical response and the ratio of stimulation current phase ³⁸. However, there has been limited in vivo research on spatial activation in RD models. Furthermore, to our knowledge, the effect of IPI on cortical spatial activation remains unexplored. The present work provides further insight into the impact of phase duration and IPI on cortical spatial activation in both healthy and retinal degenerated rats. We found that a 500 µs phase duration produces larger activation ratio in V1 compared to 1000 and 1500 µs, as demonstrated in Fig. 4(b)-(g). Additionally, our study suggested that the addition of IPI does not increase the cortical activation ratio (Fig. 7(d)-(g)). These findings imply that retinal electrical stimulation with a phase duration of 500 µs and without IPI might efficiently enhance the spatial activation of retinal implants.

This study aimed to investigate the effect of retinal electrical stimulation parameters on the visual cortical response by analyzing the activation threshold and ratio. Our findings suggest that retinal stimulation with a short phase duration (i.e., 500 µs) provides an optimal strategy for achieving the desired cortical response and threshold, without risking retinal tissue damage. These insights were observed across both LE and F1 groups, offering valuable knowledge for enhancing visual cortical response to retinal electrical stimulation in rats with retinal degeneration. However, our analysis of spatial characteristics from IPI investigation was restricted to 5 Hz electrical stimulation, and the effects of stimulation frequency on activation ratio remains unexplored. Additionally, our study indicated that higher electrical stimulation frequency results in decreased cortical response levels, yet the underlying mechanism behind this observation was not investigated in our approach and require future directions for research in this area.

6. Data availability statement

The data generated during and/or analyzed during the study are available from the corresponding author by request.

Acknowledgments

This research was supported by the Chengdu Science and Technology Program under Grant No. 2022-YF05-01360-SN, the Research Grants Council of the Hong Kong Special Administrative Region under Grant No. CityU 11207419, and the City University of Hong Kong under Grant No. CityU 7005452).

Author Contributions

H.X.: Conceptualization, Methodology, Investigation, Formal analysis, Data Curation, Writing—original draft; Z.Y.: Investigation, Formal analysis, Data Curation, Writing—original draft, Writing—review & editing; L.L.H.C.: Conceptualization, Supervision, Investigation, Resources, Writing—review & editing. All of the authors contributed to and approved the final version of the manuscript.

Competing interests

The authors declare no competing interests.

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Optimizing Electrical Stimulation Parameters to Enhance Visual Cortex Activation in Retina Degeneration Rats

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Animals

2.2 Epiretinal Electrical Stimulation Preparation

2.3 Electrophysiological Signal Recording

2.4 Impedance Measurement and Electrical Stimulation of the Retina

2.5 Characterization of Electrical Evoked Potentials of Visual Cortical Neurons

2.6 Data Processing and Analysis

3. Results

3.1 Effect of Stimulation Phase Duration and Frequency on Threshold Current and Charge

3.2 Effect of Phase Duration and Frequency on Spatial Activation in the Primary Visual Cortex

3.3 The Cortical Response Level Declined with Increasing Epiretinal Stimulation Frequency Under Equal Charge Injection

3.4 Effect of Interphase Interval on Evoked Response Amplitude in Retinal Electrical Stimulation

4. Discussion

Declarations

References

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