Optimal transcorneal electrical stimulation parameters for preserving photoreceptors in a mouse model of retinitis pigmentosa

Sam Enayati ,Karen Chang ,Anton Lennikov,Menglu Yang,Cherin Lee,Ajay Ashok,Farris Elzaridi,Christina Yen,Kasim Gunes,5,Jia Xie,Kin-Sang Cho,Tor Paaske Utheim,Dong Feng Chen,*

Abstract Retinitis pigmentosa is a hereditary retinal disease that affects rod and cone photoreceptors,leading to progressive photoreceptor loss.Previous research supports the beneficial effect of electrical stimulation on photoreceptor survival.This study aims to identify the most effective electrical stimulation parameters and functional advantages of transcorneal electrical stimulation (tcES) in mice affected by inherited retinal degeneration.Additionally,the study seeked to analyze the electric field that reaches the retina in both eyes in mice and post-mortem humans.In this study,we recorded waveforms and voltages directed to the retina during transcorneal electrical stimulation in C57BL/6J mice using an intraocular needle probe with rectangular,sine,and ramp waveforms.To investigate the functional effects of electrical stimulation on photoreceptors,we used human retinal explant cultures and rhodopsin knockout (Rho–/–) mice,demonstrating progressive photoreceptor degeneration with age.Human retinal explants isolated from the donors’ eyes were then subjected to electrical stimulation and cultured for 48 hours to simulate the neurodegenerative environment in vitro.Photoreceptor density was evaluated by rhodopsin immunolabeling.In vivo Rho–/– mice were subjected to two 5-day series of daily transcorneal electrical stimulation using rectangular and ramp waveforms.Retinal function and visual perception of mice were evaluated by electroretinography and optomotor response (OMR),respectively.Immunolabeling was used to assess the morphological and biochemical changes of the photoreceptor and bipolar cells in mouse retinas.Oscilloscope recordings indicated effective delivery of rectangular,sine,and ramp waveforms to the retina by transcorneal electrical stimulation,of which the ramp waveform required the lowest voltage.Evaluation of the total conductive resistance of the post-mortem human compared to the mouse eyes indicated higher cornea-to-retina resistance in human eyes.The temperature recordings during and after electrical stimulation indicated no significant temperature change in vivo and only a subtle temperature increase in vitro (～0.5–1.5°C).Electrical stimulation increased photoreceptor survival in human retinal explant cultures,particularly at the ramp waveform.Transcorneal electrical stimulation(rectangular+ramp) waveforms significantly improved the survival and function of S and M-cones and enhanced visual acuity based on the optomotor response results.Histology and immunolabeling demonstrated increased photoreceptor survival,improved outer nuclear layer thickness,and increased bipolar cell sprouting in Rho–/– mice.These results indicate that transcorneal electrical stimulation effectively delivers the electrical field to the retina,improves photoreceptor survival in both human and mouse retinas,and increases visual function in Rho–/–mice.Combined rectangular and ramp waveform stimulation can promote photoreceptor survival in a minimally invasive fashion.

Key Words: bipolar cells;electrical stimulation;neuroprotection;photoreceptor degeneration;retina;retinal explants;retinitis pigmentosa;transcorneal electrical stimulation;waveform

Introduction

Retinitis pigmentosa (RP) is a group of hereditary retinal diseases that primarily affects rod and cone photoreceptors,leading to progressive retinal degeneration.The main symptom of RP is night blindness,often starting in the teenage years,followed by progressive loss of peripheral vision and subsequent loss of central vision (Hamel,2006).At the cellular level,the symptoms are associated with the progressive death of the photoreceptors (Jones et al.,2016).With a prevalence rate of approximately 1 in 5000,RP is the leading cause of blindness in patients under 60 years old worldwide(Hartong et al.,2006).More than 50 genes linked to over 3000 mutations have been shown to be associated with the development of RP,producing different variants of the disease and its inheritance patterns (Daiger et al.,2013).Patients with RP often face a scarcity of viable treatment options to alleviate or postpone the advancement of their condition.Currently,the sole available RP gene therapy pertains specifically to individuals with X-linked RP attributed to mutations in the GTPase regulator,which encompasses only a subset of RP patients (Cehajic-Kapetanovic et al.,2020).

Minimally invasive microcurrent electrical stimulation (ES)is an emerging technique for treating ocular diseases and improving vision (Sehic et al.,2016).Clinical trials and animal studies have suggested that transcorneal (TcES) and transpalpebral ES can improve vision in patients with central or branched retinal arterial occlusion,dry age-related macular degeneration,RP,and other forms of retinal disease and injury(Fujikado et al.,2006;Chaikin et al.,2015;Bittner et al.,2018).ES has increased retinal outer nuclear layer (ONL) thickness,increased photoreceptor survival in Royal College of Surgeons,rats,and protected retinal ganglion cells against optic nerve axotomy-induced damage (Morimoto et al.,2007,2010).ES has been found to upregulate Müller cell production of neurotrophic factors such as brain-derived neurotrophic factor(BNDF),ciliary neurotrophic factor,insulin-like growth factor,and fibroblast growth factors (Cao et al.,1997;Morimoto et al.,2005;Seki et al.,2005;Zhou et al.,2012;Enayati et al.,2020).Additionally,ES was reported to induce direct physiological responses and increase chorioretinal blood flow,which improves the retinal microenvironment (Kurimoto et al.,2010;Sehic et al.,2016).

Mice carryingrhodopsingene deficiency (Rho–/–) are a common model of human RP,as rhodopsin mutations are observed in 25% of the autosomal dominant RP cases (Hartong et al.,2006).Rho–/–mice develop progressive photoreceptor degeneration over 3 months post-birth (Collin et al.,2020).Previous reports demonstrated the beneficial effects of the rectangular waveform ES at 100 µΑ with mixed frequencies inRho–/–mice,partly by improving photoreceptor survival (Yu et al.,2020).Two outstanding questions are whether anterior segment ES can efficiently deliver the electric field to the retina as well as the back of the eye and whether modifying the ES parameters can enhance the beneficial effects of ES.As the current cannot be measured directly in healthy human eyes,we compared ES-induced currents in the retina of the mouse and post-mortem human eyes and further determined the optimal ES parameters for improving visual function using the Rho–/–mouse model.

Methods

Animals

Rhodopsinknockout (Rho–/–) mice (n=70),male and female,6–8 weeks old,with a body weight range of 21–27 g,were originally generated by Trinity College,Dublin,Republic of Ireland.These mice are engineered with a substitution mutation in exon 2 of therhodopsingene within the C57Bl/6J strain.At 48 days of age,these mice exhibit diminished outer nuclear layer (ONL) thickness and a lack of rod ERG response.By 3 months,there is nearly complete loss of rod photoreceptors (Humphries et al.,1997).The C57BL6 mice (n=12),male and female,6–8 weeks old with body weight 23–29 g were purchased from Jackson’s Laboratory (Bar Harbor,ME,USΑ).Αll mice had no prior experimentation or drug use.Αll animals were housed mantained in specific-pathogen-free(SPF) animal facility at Schepes Eye Research Institute.The facility is accredited by American Association for Laboratory Animal Science.All animal experiments were approved by the Institutional Αnimal Care and Use Committee of the Schepens Eye Research Institute (protocol: 2020N 000219;approved on February 26,2021) and were conducted in compliance with the guidelines of the ΑRVO (Αssociation for Research in Vision and Ophthalmology,Rockville,MD,USA) statement for the Use of Animals in Ophthalmic and Vision Research in compliance with the ARRIVE guidelines (Animal Research: Reporting ofIn VivoExperiments).The mice were kept in a 12-hour light/dark cycle with free access to food and water.Genotyping ofRho–/–mice was performed with Transnetyx: Outsourced PCR Genotyping Services (www.transnetyx.com) by realtime PCR genotypic assay.At the experimental endpoint,mice were euthanized by CO2inhalation in the custom build plexiglass euthanasia chamber with a CO2fill rate of 30–70%of the chamber volume per minute according to NIH ARAC Guidelines for Euthanasia of Rodents Using Carbon Dioxide.(Shomer et al.,2020).The rodent’s death was ensured by secondary cervical dislocation after cessation of movement and respiration following CO2inhalation.

Recording of ES waveforms in the mouse retina

The Hantek2000 Oscilloscope (Hantek,Gyeonggi,Republic of Korea) was used for ES waveform and voltage recordings.Eight-week-old C57BL/6J mice were anesthetized with an intraperitoneal injection of Ketamine (100 mg/kg;Dechra Vet Products,Overland Park,KS,USΑ,383017-01) and Xylazine (20 mg/kg;Covetrus North America,Dublin,OH,USA,1XYL006).Under a surgical microscope,the positive probe was connected to a conductive (～50 Ω) 30G needle penetrating the retina,and the negative probe was connected to the negative electrode of the pulse generator STG4000(Multichannel Systems,Reutlingen,Germany),which generated ramp,rectangular,or sine waveforms at 20 Hz and 100 µΑ current.The pulse generator was set in the amplitude priority (the pulse generator adjusts the voltage settings to reach 100 µΑ current flow depending on thetissue’s resistance).The positive probe of the pulse generator was applied to the cornea through a conductive gel interface(Spectral 360;Parker Laboratories,Fairfield,NJ,USΑ).The negative electrode of the pulse generator was placed on the mouse’s abdomen through the conductive gel interface to mitigate the high resistance of the fur and the skin.Thirty recordings were acquired from each waveform and averaged.The mice were euthanized by carbon dioxide inhalation at the end of the recordings.

Human donor eyes

The Schepens Eye Research Institute Institutional Review Board reviewed the experiments conducted on post-mortem human eye material and deemed them exempt from IRB approval (REDCap ID#910).This exemption was granted as the research does not involve human subjects.The post-mortem donor eye globes were procured from Florida Lions Eye Bank(Miami,Fl,USΑ).The donors 22-03-044 (68 y.o.,female),22-03-041 (92 y.o.,female),and 21-12-054 (82 y.o.,female)without ocular pathology or known systemic diseases affecting the retina.Written informed consent was acquired from all donors in vita.The globes were received within 48 hours postmortem.Florida Lions Eye Bank is certified by the State of Florida Αgency for healthcare administration division of health quality assurance (AHCA) (https://www.fleb.org/documents/HCA22.pdf);Eye bank association of America (https://www.fleb.org/documents/ebaa_new.pdf) and Department of Health and Human Services Public Health Service Food and Drug Αdministration (FDΑ;https://www.fleb.org/documents/FDA22.pdf).All relevant procedures involving human donors and post-mortem tissue collection were conducted in adherence to theDeclaration of Helsinki.The donor eyes were utilized for post-mortem resistance recording (Figure 1) and retinal explant cultures (Figure 2).

Figure 1｜Recording of ES waveforms in the posterior segment of the eye in vivo.

Figure 2｜Electric stimulation promotes photoreceptor survival in human retinal explant cultures.

Total conductive resistance recording in human and mouse post-mortem eyes

The total conductive resistance recording of the cornea was performed as reported in the literature (Yang et al.,2022).The human donor eye recordings were performed immediately upon the globe’s arrival.For mouse post-mortem eye recordings,eight weeks old C57BL/6J mice were euthanized by carbon dioxide inhalation.The enucleated eyes were placed in the humidity chamber at 4°C for 48 hours to match the conditions and anoxia time of the human specimens.Resistances were recorded using the Hantek2000 Oscilloscope(Hantek).Αs total conductive resistance can only be measured by direct current,the conductive resistance was determined using direct current.Before each measurement,the instrument was calibrated using a 1 MΩ Resistor (E-Projects,Great Rapids,MI,USΑ,10EP5141M00).The probes were connected to the cornea’s surface (cathode probe) and to a needle probe (～50 Ω) 30G (anode probe) that was inserted into the retina.Five recordings were made per eye and averaged.

Human retinal explant cultures and electrical stimulation in vitro

Human eye globes were dissected to isolate the retina and separate it from the vitreous body.Then,under a dissection microscope,2 mm diameter biopsy punches were used to generate circular retinal explants.Three retinal explants per individual per experimental group were produced.The retinal explants were transferred onto 12-mm-diameter culture inserts (0.4 µm pore;EMD Millipore Corp.,Billerica,MΑ,USΑ) and positioned,so the photoreceptors were facing the insert membrane.The inserts were then placed into 6 well plates,and the electric current biphasic rectangular (100 µΑ 2–200 Hz,40-second cycles for 30 minutes);biphasic ramp(100 µΑ,20 Hz,30 minutes) was delivered to the cultures for 30 minutes using c-dish carbon electrode plate (Ion Optix,Westwood,MΑ,USΑ).Sham-stimulated explants were used as controls.The sham stimulation was conducted by placing the c-dish connected to the pulse generator into the cultures for 30 minutes without engaging the current flow.The explants were then flattened by gently removing the excess medium and cultured at the air-medium interface in serum-free conditions in Neurobasal-Α media (Invitrogen,Waltham,MΑ,USA,10888022) supplemented with 100 U/mL penicillin–100 U/mL streptomycin (Sigma-Αldrich,St.Louis,MO,USΑ,P4333)with no growth factors added for 48 hours to simulate the neurodegenerative environment.The explant cultures were maintained in humidified cell culture incubator HeracellTM150i(Thermo Fisher Scientific,Waltham,MΑ,USΑ) at 37°C and 5%CO2.

Rhodopsin immunostaining and quantification in human retinal explants

The cultured retinal explants were fixed with 4%paraformaldehyde at 4°C overnight,afterwards,the explants were blocked and permeabilized for 12 hours using 0.5%Triton-X in 5% normal donkey serum (D9663,Sigma-Αldrich).The explants were then incubated with a primary antibody to rhodopsin (1:200,Αbcam,Waltham,MΑ,USΑ,ab5417,RRID: ΑB_304874) at 4°C overnight.Following PBS-Tween 20 (0.05%;PBS-T) and washing (3 cycles of 15 minutes),the explants were visualized through the application of Cyanine3(1:1000,Thermo Fisher Scientific,Α10521).The cell nuclei were counterstained by incubation with 4′,6-diamidino-2-phenylindole (DΑPI) (1:5000,Sigma).Explants were gently flipped with the photoreceptor side facing up,mounted on slides with a ProLong Diamond antifade reagent (Thermo Fisher Scientific),and visualized using a fluorescent microscope Leica SP8 laser confocal microscope (Leica AG,Wetzlar,Germany).The number of rhodopsin-positive photoreceptors was counted in retinal explant images,and photoreceptor density per mm2was determined by the following equation (number of photoreceptors per area) ×image area (mm2);the results of three explants from each individual were averaged.

tcES in vivo

Six-week-oldRho–/–mice of mixed gender received two TcES sessions (tcES;n=12) or sham (n=10) treatments on one random eye under isoflurane anesthesia.TcES was performed for 5 consecutive days,with 5 days resting,followed by another 5 consecutive days of tcES).Stimulation was applied transcorneally using a conductive (～40 Ω) stainless steel probe.Two pulse generators were used in the study: STG4000(Multichannel Systems) to generate biphasic ramp waveform(100 µΑ,20 Hz,160 seconds) and Terramac (Αcuity Medical Inc.,Annapolis,MD,USA) to generate biphasic rectangular waveform (100 µΑ 2–200 Hz,40-second cycles for 160 seconds) as reported previously (Yu et al.,2020).The Terramac device was provided by Acuity Medical Inc.The conductive gel layer was placed on the cornea (Spectra 360;Parker Laboratories,Fairfield,NJ,USΑ),and the stainless steel cathode probe was applied to the gel layer without directly touching the cornea.The anode in both stimulations was placed under the mouse’s abdomen through an electrode gel interface to overcome the high resistance of the fur.Mice in the sham group were subjected to isoflurane anesthesia.The anode and cathode were similarly placed on the abdominal area and the conductive gel interface on the cornea.The stainless-steel probe made contact with the conductive gel interface on the cornea for 160 seconds without engaging the current in the pulse generator.The conductive gel application did not induce corneal epithelial toxicity or reduced tear production as reported previously(Yang et al.,2022)

Electroretinography

ERG was carried out as previously described (Yu et al.,2020).The mice were dark-adapted for 12 hours.The experiments were performed in the dark with limited 589 nm red light illumination.Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg).Tropicamide 0.5% (Sandoz,West Princeton,NJ,USΑ) was used for pupil dilatation.Mice were placed on a heating pad to maintain body temperature during testing and prevent stress cataract formation.Contact electrodes were placed centrally on both corneas.The electrodes were lubricated with eye gel(GenTeal Tears gel;Alcon,Geneva,Switzerland),the ground electrode was placed subcutaneously at the base of the tail,and a reference electrode was inserted subcutaneously at the forehead between the eyes.Rho–/–mice generally lack scotopic responses after 7 weeks of age;a photopic ERG was recorded using ColorDome LabCradle (Diagnosys,Lowell,MA,USA) inRho–/–mice and after tcES/sham treatments.

Optomotor response assessment

OMR was used to examine visual acuity (VA) inRho–/–mice after tcES/sham treatments,as we previously described using a custom-built OMR device (Shi et al.,2018.Briefly,mice were placed on a stand set surrounded by four 15.6-inch LCD monitors (Acer 16PM6Q,Acer,Schaumburg,IL,USA) and head movement was observed when presenting the mouse images of moving black and white bars.Bar width and brightness were altered increasingly until response OMR response was observed (Shi et al.,2018).The staircase paradigm was used for spatial frequency and sinusoidal gratings,with 100%contrast (black and white stripes) to measure VA.OMR was performed at 8 weeks (2 weeks after the beginning of tcES) to assess the visual function in mice.Two observers conducted data recording;mouse OMR observations were declared by both observers simultaneously.OMR observations that one of the observers did not confirm were considered false.

Retinal histology,immunohistochemistry and quantification

Cryosections were prepared as previously described(Zhao et al.,2011).In brief,mouse eyeballs were fixed in 4% paraformaldehyde at 4°C overnight,cryoprotected in 20% sucrose solution,and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek OCT Compound;Sakura,Torrance,CΑ,USΑ).Eyeballs were then sectioned into 10 µm thick slices and prepared for immunohistochemistry.Retinal sections were rinsed with PBS and incubated in a blocking buffer containing 2.5% normal donkey serum and 0.3% Triton X-100 in PBS for 30 minutes.Primary antibodies Blue-Opsin (B-opsins;rabbit,1:250,MilliporeSigma,Burlington,MΑ,USΑ,ΑB5407,RRID: ΑB_177457),Red/Green Cone Opsin,clone 7G8 (mouse,1:250,MilliporeSigma,MΑBN2492) and protein kinase C alpha PKCα (mouse,1:100,Santa Cruz Biotechnology,Dallas TX,USΑ,sc-8393,RRID:AB_628142) were incubated with the blocking buffer at 4°C overnight.Sections were rinsed three times with PBS-T and incubated with a Cy2 anti-rabbit secondary antibody(1:500,Jackson Immuno Research Laboratories,West Grove,PΑ,USΑ,111-225-144,RRID: ΑB_2338021) and Cy2 anti-mouse secondary antibody (1:500,Jackson Immuno Research Laboratories,715-225-150,RRID: ΑB_2340826) at room temperature for 2 hours.Sections were washed with PBS-T and mounted with ProLong Diamond antifade reagent(Thermo Fisher Scientific),a mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI).

Fluorescent imaging and quantification

Human retinal explants were imaged with Leica SP8 laser confocal microscope (Leica ΑG).Images of rhodopsin,PKCα,B-Opsin and Red/Green-Opsin labeling were captured on the Leica DMi8 inverted microscope equipped with a CCD camera(Leica Microsystems,Wetzlar,Germany).The thickness of the ONL was measured randomly from five or more nonconsecutive retina sections in a total of three or more mice per group.ONL was identified through the DAPI staining.The density of bipolar cell processes was determined by 16 random non-consecutive retina section images per eye that were quantified in a masked fashion.PKCα+processes that extended into the ONL were traced and quantified as positive.The opsin+cone photoreceptors were quantified from entire retina sections under the microscope in a blinded fashion.The positive photoreceptors cells in stained sections were counted manually by the “blind”observer in the aquired photographs.

Temperature recordings

We conducted temperature recording in bothin vivoandin vitrosettings to evaluate the potential heat generation during ES.We measured temperature changes using an infrared thermometer PCE-895 (PCE Instruments,Jupiter,FL,USΑ) with 0.1°C resolution.In vitrostimulations were conducted using the c-plate (Ion Optix,Westwood,MΑ,USΑ).The 6-well plates were filled with 2 mL of culture media Neurobasal-A media(Invitrogen,10888022) per well,and c-plate electrodes were inserted into the wells.The plate was left for 1 hour at room temperature to equilibrate.Then electric current biphasic rectangular,biphasic ramp,and biphasic sine were delivered to the cultures for 30 minutes using a c-dish carbon electrode plate (Ion Optix,Westwood,MΑ,USΑ).The temperatures of the media were detected at the end of the experiment.The temperature of the media without ES was used as the control.The ambient temperature was monitored and remained within 0.1 C variation during the 30-minute experiment.Forin vivoexperiments,we stimulated the mice with (Ramp,100µΑ,20 Hz,160 seconds) with a metal probe electrode while monitoring the temperature change of the cornea using the measurements before stimulation as the control.

Statistical analysis

Experimental values were expressed as the mean ± SD for the respective groups.Statistical analyses were performed with GraphPad Prism software (version 10.0.0,https://www.graphpad.com/scientific-software/prism/).The unpairedt-test was used to compare the two groups,and one-way analysis of variance with Tukey’s multiple comparisons was used when comparing multiple groups.Α value ofP<0.05 was considered statistically significant.

Results

Current waveforms and electrical field penetration following transcorneal ES in post-mortem mouse and human eyes

To determine if TcES reaches the posterior eye or retina,we recorded the current,waveforms,and voltages in the subretinal space using an oscilloscope.The oscilloscope’s needle probe (50 Ω) was inserted into the subretinal space of an anesthetized mouse (Figure 1A,left).tcES at a biphasic rectangular,sine,or ramp waveform was delivered at 20 Hz,a frequency previously shown to be optimal for retinal cells(Enayati et al.,2020;Yu et al.,2020).The corresponding output waveform was recorded following all ES (Figure 1B–D).The output voltage needed to reach a value of 100 µΑ current in the subretinal space of anesthetized mice varied significantly among different waveforms (Figure 1E).These results suggest that the ramp waveform requires the least voltage to reach the retina.To test if the ES current flow assessed in the mouse eye can be translated to humans,we compared the tcESinduced currents and conductive resistance in the mouse and donor human eyes.The conductive resistance of post-mortem mouse and human eyes was measured by placing multimeter probes on the cornea and a needle in the subretinal space(Figure 1A,right).The transcorneal-retinal resistance measured from the post-mortem human eye was three times higher than that measured in the post-mortem mouse eye(Figure 1F).Based on this data,approximately three times the tcES voltage used in mouse eyes is required to achieve the same level of tcES current in human eyes.

Ramp waveform ES is more effective at improving photoreceptor survival than rectangular ES in human retinal explant cultures

We next compared the neuroprotective potential of different ES waveforms in human post-mortem retinal explant cultures.Previous studies suggested that employing ES at a ramp waveform (20 Hz) or a series of rectangular pulse trains at increasing frequencies from 20–200 Hz improved retinal cell survivalin vitroandin vivo(Enayati et al.,2020;Yu et al.,2020).It prompted us to evaluate the effects of these ES conditions on photoreceptor cell survival in culture.Using post-mortem human retinal tissue explants.The graphical summary of the experiment is presented inFigure 2A.Shamstimulated explants served as the controls.Following 48 hours of incubation after ES,the survival of photoreceptors was assessed using rhodopsin immunostaining.In control cultures,substantial loss of the photoreceptors was observed,with only a few rhodopsin+cells detectable (Figure 2B).Rectangular ES improved the number of surviving photoreceptors (Figure 2C),but this number did not reach significance (P>0.05).Remarkably,the stimulation with ramp waveform resulted in dramatically increased photoreceptor survival after 48 hours of culture (Figure 2D).Quantitative analysis of the rhodopsinpositive cells in the explants (Figure 2E) indicated that ramp waveforms have a significant increase in the number of rhodopsin-positive surviving photoreceptors when compared with control (P<0.05) or rectangular waveform (P<0.05).These results suggest the potent neuroprotective effects of ramp waveform ES.

Transcorneal ES improves retinal function and visual behavior in Rho–/– mice

To determine the functional benefits of tcESin vivo,we assessed visual perception measured by the OMR-based assay and quantified photoreceptor survival (Shi et al.,2018).Using the previously demonstrated ES paradigm with a current of 100 µΑ series of rectangular pulse trains with increasing frequencies from 20–200 Hz (Yu et al.,2020),we performed tcES inRho–/–mice.Following two 5-day sessions starting at 6 weeks of age (Additional Figure 1A),we did not observe a significant enhancement of visual acuity inRho–/–mice (Additional Figure 1B;P>0.05).We hypothesized that ES with combined rectangular and ramp waveforms might present a more potent stimulation paradigm invoking neuroprotective responses and improving retinal and visual function inRho–/–mice.We employed tcES with a combined ramp waveform (100 µΑ,20 Hz,160 seconds) followed by stimulation with the rectangular waveform of (100 µΑ,2-200 Hz,40 seconds/cycle,160 seconds) or sham treatments to one random eye ofRho–/–mice,starting when mice reached 6 weeks old.Following two sessions of 5 consecutive days of tcES with a 5-day-rest in between (Figure 3A),we noted that mouse eyes that received combined tcES treatments exhibited significantly improved retinal cone function,as measured by ERG,than those in sham-treated mouse eyes(Figure 3B).Significant enhancement of M-cone (Figure 3C)and S-cone (Figure 3D) functions was detected 2 weeks after the first ES treatment (P<0.05) when compared with sham stimulation.Moreover,we observed significant (P<0.05)improvement in visual acuity in ES-treated eyes compared to sham-treated eyes (Figure 3E).These results indicate the functional benefits of tcES with combined waveforms of rectangular and ramp series.

Figure 3｜Combined rectangular and ramp tcES improves ERG and OMR responses in Rho–/– mice.

tcES attenuates photoreceptor loss in vivo

We counted photoreceptor cells in retinal sections to determine if tcES improves retinal and visual functions by preserving photoreceptors.Immunostaining revealed that tcES ameliorated the loss of B-opsins (Figure4A) and Red/Green-opsins cells (Figure 4B).Quantitative analysis of B-opsins positive cells (P<0.01;Figure 4C);Red/Green-opsins positive cells (P<0.05;Figure 4D) and total opsin positive cells numbers (P<0.05;Figure 4E) demonstrated significantly improved survival of B-opsin,R/G-opsin,and total Opsinpositive cells in the tcES group compared to sham-treated contralateral eyes).ONL thickness was also significantly increased (P<0.001) in tcES-treated retinas compared to sham-treated eyes.This indicates improved survival of all types of photoreceptor cells after tcES treatment (Figure 4F).These results indicate tcES ameliorated photoreceptor loss in Rho–/–mice retina.

Figure 4｜tcES increases the survival of photoreceptors.

tcES increases the density of bipolar cell sprouting and preserves the retinal outer nuclear layer

Bipolar cells have been shown to undergo structural changes and neurite sprouting that contribute to retinal function and recovery (Lewis et al.,1998).To evaluate if tcES affected bipolar cell plasticity,we performed immunolabeling for the bipolar cell-specific marker Protein kinase Cα (PKCα).We observed prominent upregulation of PKCα immunolabeling in the tcES-treated retina,primarily associated with bipolar cell processes,compared to the sham-treated retina(Figure 5AandB).We also noted an increase in bipolar cell processes that grew into the ONL.Quantitative analysis of the density of the bipolar cell dendrite extending into the ONL confirmed that tcES significantly increased the density of bipolar cell sprouting (P<0.001) into the ONL compared to the sham-treated group (Figure 5C).Increased retinal plasticity and bipolar cell sprouting in response to the tcES may be an additional mechanism contributing to increased photoreceptor survival and function in theRho–/–mice.

Figure 5｜tcES increases bipolar cell sprouting.

Temperature change in vivo and in vitro during electric stimulation

Temperature recordings were conductedin vivoandin vitro.In vivoexperiments involving mice did not reveal any significant temperature changes during the 160-second stimulation session (Additional Figure 1C).In contrast,in vitroexperiments showed that after 30 minutes,the media temperature increased slightly by 0.5°C during the ramp waveform current,but this increase was not significantly different (P>0.05) from that of the control plate.Similarly,the rectangular ES showed no significant difference (P>0.05) from the control plate.However,the sine waveform significantly (P<0.001) increased the media temperature,which rose by 1.5°C compared to the control (Additional Figure 1D).Based on these results,we can conclude that microcurrent ES produces no discernable tissue heatingin vivoand minimal heating effectsin vitro.

Discussion

This study presented evidence demonstrating effective penetration of electric field reaching the subretinal space following tcES.Among the three waveforms tested,the ramp waveform was most efficient in penetrating the mouse and human eyes and improving photoreceptor survival in human retinal explant cultures.Combining the ramp waveform and the rectangular waveform tcES enhanced photoreceptor survival and visual functions in Rho–/–mice.These results defined a unique and optimized regimen of tcES for rescuing photoreceptors and visual function in RP.

Clinical trials have shown that tcES improves vision in some patients with RP (Chow et al.,2004;Kurimoto et al.,2010;Wang et al.,2011;Yin et al.,2016).The most effective waveform and settings of ES remain unclear,with the field of preclinical and clinical studies predominantly employing monophasic or biphasic rectangular pulses at varying frequencies (Sehic et al.,2016).In other studies,such as that reported by Jolly et al.(2020) that delivered to RP patients a biphasic rectangular waveform of 5 ms pulse duration at 20 Hz and 20–1000 µΑ current amplitude (until the patients reached the phosphene threshold),no significant improvement in visual function was noted.Similarly,we noted that although ES at a rectangular waveform significantly improved photoreceptor survival and function as assessed by ERG,it did not enhance visual perception as measured by the OMR response inRho–/–mice (Yu et al.,2020).Whereas,in the present study,we showed that a ramp waveform had unique advantages in promoting retinal neuron survival and function.Intriguingly,the ramp waveform requires the least voltage to reach the posterior eye,suggesting its high efficiency and low impedance in delivering electrical field.Similar results were recently reported by Navntoft et al.(2020)where the ramp waveform requires less voltage potential to achieve the current amplitude needed to evoke responses in cochlear neurons with ramp waveforms similar in amplitude to responses obtained with the rectangular waveformin vivo.In human retinal explant cultures,we showed that ramp waveform ES is far more effective at promoting photoreceptor cell survival than rectangular waveform.This is in line with thein vivoobservation inRho–/–mice that ramp,but not rectangular,ES enhanced photoreceptor survival and VA assessed by OMR.It is worth noting that photoreceptor degeneration in cultured human retinal explants was far more severe and acute than inRho–/–mice.This serves as a highly likely explanation for the even less potent neuroprotective effect of rectangular ES in explant cultures than inRho–/–micein vivo.

Understanding the ES safety threshold is critical for therapeutic purposes,as it sets the maximal amount of electrical energy that can be applied to produce therapeutic benefits without causing tissue damage.We were tempted to address this question in the animal model by comparing the electric current that reached the retina in the corpus mouse and human eyes.In the case of ES in anesthetized mice,we did not observe any tissue heating,skin defects,or erosions following 4-minute biphasic ESin vivo,even after repeated stimulations.Ourin vitrostudy found that prolonged stimulation with biphasic waveforms for 60 minutes with amplitudes exceeding 500 µΑ resulted in a minor but significant increase in cell toxicity (Lennikov et al.,2022).In agreement with the clinical observations (Rizzo et al.,2003;Cogan et al.,2016),these findings suggest that microcurrent stimulation with amplitudes under 500 µΑ is generally safe and does not cause any apparent adverse effects.

Our resistance recordings between the corneal surface and subretinal space suggest that the mouse eye may present a translatable model for evaluating ES settings,with the mouse eye showing conductive resistance at about～3 times less than the human eye when measured between the cornea and subretinal space.The amplitude priority ES would not be affected by the difference in the resistance as the voltage values would be adjusted by the pulse generator to reach the target amplitude;however,voltage priority ES settings that are common in clinical trials have to be scaled accordingly to accommodate for higher resistance of the human cornea.While we measured the conductive resistance of the eye in corpus human eyes,the post-mortem tissues with 48 hours of anoxia time may display different conductive resistance and capacitance parameters than live tissues.

Patient compliance,especially in outpatient settings,is reported to be between 40% and 50% for long-term medication therapies (Jin et al.,2008).Thus,an effective ES therapy must maintain its beneficial effects even with treatment interruptions.In vivoexperiments demonstrated that a 5 consecutive days ES treatment regimen and a 5-day rest significantly improved visual outcomes.The results are promising as they suggest that even interrupted ES can benefit an RP condition significantly.Further studies on how long the beneficial effects of the ES last after the end of the stimulation and how often ES should be applied are currently underway.Several potential mechanisms may be involved in ES’s therapeutic effects on retinal and photoreceptor degenerative diseases.One possible mechanism is the increased production of growth factors such as basic fibroblast growth factor (Αrias-Calderon et al.,2023) and BDNF (Kimura et al.,2019).These factors are known to play a critical role in promoting the survival and regeneration of retinal neurons,including photoreceptor cells.ES has been shown to stimulate the release of basic fibroblast growth factor and BDNF (Di Pierdomenico et al.,2018;Valiente-Soriano et al.,2019).ES is also shown to improve retinal blood flow circulation,enhancing the delivery of oxygen and nutrients to photoreceptor cells (Liu et al.,2022).In addition,this increased blood flow may also promote the removal of metabolic wastes or products from the retina to enhance the survival and function of photoreceptor cells (Kurimoto et al.,2010;Sehic et al.,2016).Interestingly,our temperature recordings indicated that microcurrent ES produces negligible tissue heating,suggesting that the therapeutic effects of ES are unrelated to heat but a direct response to ES.

The findings in the current study also indicate that tcES promotes retinal plasticity and increases the sprouting of bipolar cells in the retinas ofRho–/–mice.PKCα is a marker of bipolar cells (Greferath et al.,1990),and its antibody labels their dendritic processes and synaptic terminals (Kolb et al.,1993).PCKα activation requires an increase in intracellular Ca2+and binding to diacylglycerol,which leads to a transfer of PKCα to the cell membrane and potentiates the release of neurotransmitters at the synaptic site (Berglund et al.,2002).PKCα activation reduces GΑBΑ-induced currents in isolated rat bipolar cell cultures (Karschin and Wässle,1990).Our finding is in line with the work by Haq et al.(2018) that ES benefits degenerating photoreceptors by enhancing synaptic interactions through increasing calcium signaling,glutamate and GΑBΑ expression,and the gap junction-coupled neuronal networks that facilitate the signaling between photoreceptors and retinal ganglion cells.

RNΑ sequencing analyses in conventionally labeled “electrically non-responsive”cells,such as Müller cells (Enayati et al.,2020) and mouse microglial cell line BV-2 cells (Lennikov et al.,2022) revealed that ES’s effect is not limited to only neuronal cells.ES increased synaptic pathway genes,such as cholinergic and GABAergic signals,in Müller cells (Enayati et al.,2020).Increased neurotransmission between cells or sprouting from adjacent neurons may improve photoreceptor communication via bipolar cells to retinal ganglion cells (Boccuni and Fairless,2022).Furthermore,ES reduced proinflammatory activation,phagocytosis,and migration capacity in microglia,likely associated with its effects on Ca2+signaling,impacting cell motility and metabolism-related genes (Lennikov et al.,2022).Ozaki et al.(2022) reported that activation and migration of microglia towards the photoreceptor layer are associated with the progression of photoreceptor degeneration inRho–/–mice.In a different model of retinal degeneration Rd10 mutant mice,the loss of photoreceptors was strongly associated with microglial phagocytosis of living photoreceptors due to the presence of “eat-me”signals in afflicted cells.A similar process is likely to occur inRho–/–retina;thus,decreased phagocytosis,motility,and proinflammatory activation induced by ES may underlie the beneficial effects of ES on photoreceptors’ survival.Recently,Yang et al.(2022)presented results that cornea-to-retina resistance is higher than orbital skin-to-retina,suggesting even higher efficacy of the electric field delivery and less discomfort to the patients in clinical use.The dry eye symptoms reported in clinical studies of TcES (Jolly et al.,2020) are explained by the TcES inhibiting both transmembrane and secretory mucin production,where electrical current evoked by transpalpebral bypasses the cornea and does not induce such effects (Yang et al.,2022).

Conclusion

This study shows that tcES ameliorates retinal neurodegeneration and improves vision inRho–/–mice.Low current ES with combined rectangular and ramp waveforms(100 µΑ,20 Hz,160 seconds,followed by 100 µΑ 2–200 Hz,40-second cycles for 160 seconds) presents an optimal stimulation that promotes photoreceptor survival and function,increases the sprouting of bipolar cells,and improves mouse visual perception.The ramp waveform requires the least voltage potential to reach the posterior segment of the eye and demonstrates a more prominent neuroprotective effect in human retinal explant cultures when compared with rectangular currents.These results identified a unique ES waveform that is effective at rescuing photoreceptors in the RP model and suggested the possibility of applying minimally invasive tcES or,in the case of transpalpebral,non-invasive treatment for ameliorating vision loss in patients with RP.

Acknowledgments:The authors would like to acknowledge Mr.Irvin Yi and Ms.Julie Chen(Schepens Eye Research Institute of Massachusetts Eye and Ear,Department of Ophthalmology)for proofreading the manuscript and“blind”quantification.Mellissa A.Pottinger(Recovery Technician Florida Lions Eye Bank,Bascom Palmer Eye Institute,University of Miami Miller School of Medicine)for assistance with procurement of human donor eyes.Figures 1A and 3A,and Additional figure 1A were created using BioRender.com.The rhodopsin knockout mice are a gift from Michael Young’s lab at Schepens Eye Research Institute that was originally developed by Dr.Peter Humphries and his team at Trinity College,Dublin.

Author contributions:SE,KC,KSC,TPU,and DFC conceived and designed the study.MY and AL conducted ocular posterior segment waveform,voltage,and resistance recordings.AL,AA,CY,and CL conducted retinal explant experiments.SE,KC,AL,KSC,CL,CY,FE,KG,and JX conducted in vivo and ex vivo experiments and evaluations.SE,KC,AL,AA,MY,CY,TPU,and DFC wrote the manuscript.KSC,TPU,and DFC critically revised the manuscript.All authors reviewed and approved the final version of the manuscript.

Conflicts of interest:DFC is a consultant of I-lumen Scientific.DFC,AL,KC,KSC,and TPU are inventors of a patent application for using ES technology for treating eye diseases.The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Acuity Medical Inc.provided the ES device and has no knowledge or influence on the study design,results,or conclusions.

Data availability statement:All relevant data are within the paper and its Additional files.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional file:

Additional Figure 1:Transcorneal electrical stimulation(tcES)with only biphasic rectangular current at increasing pulse frequencies does not improve OMR responses in Rho–/– mice.