Retinal degeneration (RD) diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are inherited or acquired neurodegeneration disorders that involve progressive deterioration of the retina tissue. Most importantly, RP in humans involves the progressive structural and functional loss of light-sensitive outer retinal neurons (photoreceptors) and this eventually culminates in blindness. Photoreceptor loss in RD has been reported to induce gradual retinal remodeling however, the survival of bipolar cells and retinal ganglion cells has been demonstrated in the inner retina. This survival phenomenon has sparked a light of hope in the research community and to date scientific research into potential therapeutic approaches for the restoration of partial or full vision are still being conducted.

For several decades, electrical stimulation of biological organs and tissues has been paving new paths for the treatment of neurodegenerative and neurological diseases including chronic pain, Parkinson’s disease, hearing loss and blindness. Implantable bionic vision devices such as retinal prostheses function on the principle of electrical stimulation of the surviving inner retinal neurons with the sole purpose of activating the visual centers of the brain and ultimately restoring visual perception. The use of bionic vision devices has been reported to provide significant improvements to visual perception in the blind population. Despite this, bionic vision device users need to habitually turned on their visual implants for almost 16 hours period to aid their daily activities. Consequently, this routine will unavoidably trigger neuromodulation of neural activities in both visual and non-visual brain regions. Most studies on invasive and non-invasive vision restoration methods only report the effect of electrical stimulation parameters on the efficacy and safety of the stimulating electrode and the tissues in close proximity to the stimulation site. However, the potential effect of the prolonged electrical stimulation of the retina on the brain electrophysiology has not been comprehensively studied in the blind population. In previous studies, stimulation of the motor cortex neurons via magnetic and electrical methods has been reported to cause neuro-cortical modulation with long lasting after-effects on brain circuits. Therefore, it is of great relevance to study brain neural network activities and brain responses to both short-term and long-term electrical stimulation of the retina under varying parameters of stimulation. The results reported in the present study will provide clear-cut evidence for activation of the primary visual center of the retinal degeneration brain. Additionally, data presented in this study will provide for the first-time electrophysiological knowledge about how non-invasive retinal electrical stimulation neuromodulates cognitive indices of the RD brain and explore whether such neuromodulation has long lasting effects in both visual and non-visual areas of the RD brain.

In the present study, retinal degeneration 10 (rd10) mice were used as animal models for the human RP disease. Silver wire of diameter 0.4 mm, surface area 0.13 mm² and impedance 0.143 ± 0.014 kΩ (in 0.1 M phosphate buffered saline) was used as the stimulating electrode to stimulate the right corneal surface of the rd10 mice via a non-invasive method called prolonged transcorneal electrical stimulation (pTES). Subsequently electrocorticography (ECoG) was employed to record resting state or spontaneous neural signals from the non-visual (prefrontal cortex) and visual (primary visual cortex) regions in both hemispheres of the brain (left hemisphere or contralateral region and right hemisphere or ipsilateral region).

As a first step, we studied the effect of varying the electrical stimulation frequency on the ECoG responses in awake and anesthetized brain states of RD mice. Absolute power, functional connectivity (coherence) and feedforward directional connectivity (normalized symbolic transfer entropy) were used to characterize cortical neural excitation responses in awake and anesthetized states of rd10 mice following pTES [30 min/day (for 7 days)] with 2 ms/phase charge-balanced biphasic pulses and stimulation current of 400 µA (charge: 0.8 µC) at varying frequencies (2 Hz, 10 Hz and 20 Hz). The results revealed that pTES is able to modulate the resting state brain activity of rd10 mice in a stimulation frequency and brain state dependent manner respectively. In this regard, the awake brain state was found to be more responsive to the effects of pTES with 10 Hz stimulation frequency producing increased neural activity and feedforward directional connectivity in theta, alpha and beta oscillatory bands following pTES compared with unstimulated controls. Again, this experiment demonstrated significant maintenance of the increased feedforward directional connectivity in theta, alpha and beta oscillations long after the end of the stimulation period.

Previous research has reported the involvement of broadband gamma and narrowband gamma oscillations in excitatory and inhibitory network transmission. Taking this into account, we then examined neuromodulatory alterations in functional and effective connectivity indices between the prefrontal cortex and the primary visual cortex by varying the electrical stimulation pulse duration at 0.5 ms/phase, (0.2 µC), 2 ms/phase (0.8 µC) and 5 ms/phase (2 µC). The results revealed that a short pulse duration of 0.5 ms/phase strongly enhanced and maintained the increase in coherence and directional connectivity of broadband and narrowband gamma oscillations between the contralateral primary visual cortex and contralateral prefrontal cortex of rd10 mice compared to the unstimulated controls.

Next, we aimed to study the impact of stimulation current amplitudes on cognitive neural synchrony during different stages of transcorneal electrical stimulation (TES) in rd10 mice. To achieve this aim, we varied the stimulation current amplitude of TES at 400 µA (0.8 µC), 500 µA (1 µC) and 600 µA (1.2 µC) while keeping the stimulation frequency and biphasic pulse duration constant at 10 Hz and 2 ms/phase respectively. Subsequently we analyzed alterations in functional and directional connectivity indices of coherence, cross-frequency coupling, and directional connectivity in both contralateral primary visual cortex and the contralateral prefrontal cortex. The results, showed that transient TES was not sufficient to alter the precepts of brain coherence and connectivity. However, following pTES (post-stimulation stage 1), we identified enhanced increase in theta-gamma cross-frequency coupling. Meanwhile enhanced coherence and directional connectivity appeared predominantly in theta, alpha and beta oscillations. These alterations observed in both recorded brain regions were mostly dependent on the current amplitude of retinal stimulation. Interestingly, long after the end of the pTES (post-stimulation stage 2) we observed sustained increase in network coherence and connectivity patterns at the level of cross-oscillatory interaction, functional connectivity and directional inter-regional communication between the primary visual cortex and prefrontal cortex.

Last, we aimed to investigate the safety profile of pTES in the inner retinal neurons (RGCs and microglial cells) of mice with intact vision. We performed pTES on the right eye of normal-sighted C57/BL6 mice at varying current amplitudes (400 µA, 500 µA and 600 µA), high stimulation frequency (20 Hz) and high biphasic pulse duration (5 ms/phase). Afterwards we performed immunohistochemistry techniques to evaluate the safety profile of pTES in the inner retinal neurons (RGCs and microglia). Our results revealed that pTES was well tolerated in normal-sighted C57BL/6 mice which maintained retinal integrity and no observable difference between the RGC and microglia counts respectively across all stimulation groups compared with their corresponding sham control groups (left eyes without pTES). From our experiment, we concluded that pTES in the normal-sighted C57BL/6 mice presents no adverse effects to the investigated inner retinal neurons and thus pTES is a useful non-invasive stimulation technique with excellent safety profile.

In summary, from all the results reported in the aforementioned experiments of the present study, the following conclusions are suggested. Firstly, the awake brain is more responsive to the effects of pTES. Secondly, a short pulse pTES is necessary to enhance narrow band and broad band gamma oscillations which have previously been reported to play key roles in cortical excitatory and inhibitory responses. Thirdly, pTES causes sustained neuromodulatory effects in both the visual region and the non-visual brain region by altering electrophysiological cognitive indices such as functional and directional connectivity measures depending on the parameters of stimulation current amplitude. Fourthly, as a non-invasive stimulation technique, pTES is well tolerated and presents no adverse effects to the histological integrity of the investigated retinal neurons in normal sighted C57BL/6 mice.

The core implication of the present study findings is that it provides great insight for which researchers in the field could safely apply a non-invasive stimulation paradigm, specifically pTES as a novel neuromodulatory approach to target both visual and non-visual regions of the brain directly from the mammalian eye.