Electrical stimulation alters DNA methylation and promotes neurite outgrowth

Abstract

Electrical stimulation (ES) influences neural regeneration and functionality. We here investigate whether ES regulates DNA demethylation, a critical epigenetic event known to influence nerve regeneration. Retinal ganglion cells (RGCs) have long served as a standard model for central nervous system neurons, whose growth and disease development are reportedly affected by DNA methylation. The current study focuses on the ability of ES to rescue RGCs and preserve vision by modulating DNA demethylation. To evaluate DNA demethylation pattern during development, RGCs from mice at different stages of development, were analyzed using qPCR for ten-eleven translocation (TETs) and immunostained for 5 hydroxymethylcytosine (5hmc) and 5 methylcytosine (5mc). To understand the effect of ES on neurite outgrowth and DNA demethylation, cells were subjected to ES at 75 μAmp biphasic ramp for 20 min and cultured for 5 days. ES increased TETs mediated neurite outgrowth, DNA demethylation, TET1 and growth associated protein 43 levels significantly. Immunostaining of PC12 cells following ES for histone 3 lysine 9 trimethylation showed cells attained an antiheterochromatin configuration. Cultured mouse and human retinal explants stained with β-III tubulin exhibited increased neurite growth following ES. Finally, mice subjected to optic nerve crush injury followed by ES exhibited improved RGCs function and phenotype as validated using electroretinogram and immunohistochemistry. Our results point to a possible therapeutic regulation of DNA demethylation by ES in neurons.

1 |. INTRODUCTION

Glaucoma is the leading cause of irreversible blindness due to the damage to the optic nerve (ON) and death of retinal ganglion cells (RGCs).¹ The limited regenerative potential of RGCs and their axons poses a downside in restoring vision.²

Recently, the field of epigenetics has received much attention in the wake of discoveries of epigenetic factors that influence disease progression and the functions of cellular regenerative machinery.^3–5 Epigenetics modifications, such as DNA and histone methylation and demethylation, play a vital role in RGC homeostasis, regeneration, and protection.^3,6 Most of the factors and enzymes that fuel these epigenetic events have only been emerging in the past few decades. Among these factors, ten-eleven translocation (TET) protein family are mainly responsible for DNA demethylation and several studies report that their expression is important for ocular tissue homeostasis and regeneration.^5,7,8 A recent prominent study showed that vision restoration is TET1/2 dependent, following ectopic expression of Oct4 (also known as Pou5f1), Sox2, and Klf4 genes (OSK) in RGCs.⁴ Additionally, TET1-dependent deletion of PTEN was a proven regenerative event in an optic nerve crush (ONC) model.⁹ TET knockout results in inefficient myelin repair and axo-myelinic swellings which also alters astrocyte morphology and impairs neuronal function.^5,10 Therefore, any strategy to increase DNA demethylation via improving TET(s) expression can plausibly reconfigure DNA-histone structure to be translationally active and neuroprotective. Epigenetic related enzymatic including TET expression can be modulated by influencing the environment. One proven strategy that can help with this approach is the application of microcurrent electrical stimulation (ES).

ES is currently being explored across various research areas to understand its beneficial effects.^11–14 It has proven its efficacy in regenerative medicine research and studies have also shown that it has the potential to regulate epigenetic events.¹⁵ In a recent study, nanosecond pulsed electric fields was shown to cause downregulation of DNA methylation transferase 1 (DNMT1), which mediated the DNMT1-OCT4/NANOG axis resulting in the differentiation potential of mesenchymal stem cells.¹⁵ The beneficial effect of ES in ocular pathologies have been reported in retinitis pigmentosa,¹³ age-related macular degeneration,¹⁶ and retinal arterial occlusion.¹⁷ Recent studies from our lab have also shown that ES induces retinal Müller cell proliferation, photoreceptor survival, and mitigates microglial activation in the retina.^18–20 However, the underlying molecular machinery that promotes ES induced therapeutic effects in the eye remains to be elucidated.

The aim of the present study is to examine whether electric current has the potential to improve RGCs regeneration and functionality by modulating the epigenetic status especially that of DNA demethylating factors. Our data suggest that ES alters the levels of epigenetic factors that drive the transcription program of pro-regenerative genes in axotomized RGCs in vitro and in vivo.

2 |. MATERIALS AND METHODS

2.1 |. PC12 cell culture

PC12 cells (neuron-like attributes) (CRL-1721, ATCC—The Global Bioresource Center, Manassas, VA, USA) were cultured using RPMI supplemented with 5% fetal bovine serum, 10% horse serum, and a mixture of 1% of penicillin/streptomycin and incubated at 37°C in a humid 5% CO₂ environment. All cell culture plates/flasks were coated with poly-d-lysine (Sigma). Cells were induced to differentiate into neurons by administrating nerve growth factor (NGF) (Sigma-Aldrich N6009-4X25UG) at 100 ng/mL.²¹ However, experiments carried out to analyze the neurite outgrowth efficacy of the treatments, used 5 ng/mL of NGF in addition to the standard 100 ng/mL. TETi76 diethyl ester (SML3121-Millipore Sigma), a pan TET inhibitor was used at 12.5 μM (16 h treatment) to block TET activity in PC12 cells.

2.2 |. Primary RGC isolation

Primary mouse RGCs were isolated from the retinas of embryonic 14 days old (E-14), postnatal day 0 (P-0) and 3-month-old C57BL/6J mice (Jacksons Laboratory).²² In brief, retinas were dissected in Neurobasal-A medium (Gibco) and immersed into the pre-warm papain solution (Papain Dissociation System; Worthington Biochemical Corp.) at 37°C for 5 min. Equal volume of inhibitor solution (Papain Dissociation System; Worthington Biochemical Corp.) was added to cease the papain digestion. Cells were centrifuged at 300 RCF for 10 min and re-suspended in 500 μL of buffer solution containing rinsing solution (MACS Cell Separation system; Miltenyi Biotec) with 0.5% Bovine serum albumin (BSA) (1:20; BSA stock solution; Miltenyi Biotec), and incubated with 30 μL of Thy-1.2 antibody conjugated micro-magnetic beads (MACS Cell Separation system; Miltenyi Biotec) for 15 min at 4°C. The cell suspension was loaded onto pre-wet magnetic columns with 30 μm pre-separation filters (MACS Cell Separation system; Miltenyi Biotec), followed by washing with buffer solution. RGCs were eluted using 1 mL buffer solution after the magnetic field was removed.

2.3 |. Human eye globes

Human cadaver eye globes were acquired from Lions Eye Bank (Florida). Donors ranged in age from 68 to 92 years (Table 1). All experiments involving human tissue were performed in compliance with the tenets of the Declaration of Helsinki.

2.4 |. Animals

B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy-1 YFP)²³ and C57BL/6J mice were bought from Jackson Laboratory. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Schepens Eye Research Institute and followed the standards of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The mice were kept in a 12-h light/dark cycle with free access to food and water. At the experimental endpoint, mice were euthanized by CO₂ inhalation and secondary cervical dislocation.

2.5 |. ONC injury

ONC was carried out using established protocol.^24,25 Six-week-old mice were anesthetized with a mixture of ketamine and xylazine. The ON from one eye was exposed following surgery and crushed for 5 s using fine forceps. Subcutaneous injection of buprenorphine (Ethiqa XR) for pain management. After 24 h of recovery, mice were divided into two groups: one group received ES (procedure explained in later section), and the other group received a sham procedure with no ES in the ON crushed eye globe. After euthanization of mice, ONs were collected, preserved with 4% paraformaldehyde (PFA) and sectioned. Nerve sections were further stained with antibodies (Table 2) and imaged under the fluorescence microscope.

2.6 |. Electric stimulation in cell cultures, explants, and animals

Optimal ES conditions were optimized based on earlier reports published by our group.^17,18 The electric stimulation was performed in all experiments using STG4000 (Multichannel Systems) pulse generator. For in vitro experiments, ES with a biphasic ramp waveform at 75 μAmp and 50 ms pulse duration was applied for 20 min. The electric current was delivered to cultures and explants using a c-dish carbon electrode plate (Ion Optix) modified for alternating current circuits. Human and mouse retinal explants were collected and cultured using trans-well membrane (0.4 μm pore-Corning, Falcon) coated with Corning^® Matrigel^® Growth Factor Reduced Basement Membrane Matrix and RGC culture medium²⁶ for 7 days and ES (biphasic RAMP-150 μAmp-50 ms-20 min) was delivered on days 0, 3, and 5. On day 7, the tissues were immunostained for β-III tubulin to reveal the neurites. A 2 mm biopsy punch was used for human retinal explant preparation. For mice retinal explants, uniform equisized retinal sections from the same area in the retina were cultured and analyzed with and without exposure to ES. Uniformly sized retinal explants were cultured for 24 h after ES. TET1 messenger RNA (mRNA) levels were assessed by qPCR. Non-electrically or sham-stimulated (Non-ES) samples were used as controls in all experiments.

Noninvasive ES in vivo was conducted in B6.Cg-Tg (Thy1-YFP)16Jrs/J (Thy-1 YFP) mice anesthetized using isoflurane, 24 h after ONC. A conductive electrode gel (Spectral 360; Parker Laboratories) was applied around the eyelids to facilitate electrical conductivity and reduce conductive resistance. The electrode probe was placed on four equi-distanced spots around the mouse orbit for 5 min/spot. ES at a biphasic ramp waveform (150 μAmp with 50 ms pulse duration) was applied for 20 min in each mouse. The ground electrode was placed at the mouse abdomen. ES was conducted every other day starting 24 h after ONC for a period of 4 weeks. Mice received ONC and sham stimulation were served as controls.

2.7 |. Electroretinography

To study the functional effect of ES in the ONC model, electroretinogram positive scotopic threshold response (pSTR) were recorded.²⁷ Mice were dark-adapted overnight and anesthetized with an intraperitoneal injection of ketamine (120 mg/kg)/xylazine (20 mg/kg). Pupils were dilated using 0.5% tropicamide and placed on a 37°C warming pad in a Ganzfeld bowl (Diagnosys LLC) throughout the recording. Electroretinographs of both eyes were recorded simultaneously with two contact electrodes lubricated with GenTeal gel (Novartis), placed centrally on the corneas. The ground electrode and reference electrode were inserted subcutaneously at the base of the tail and forehead, respectively. The pSTR wave amplitude was recorded (light intensity = 2.33 × 10⁻⁵ cd.s/m²) and plotted graphically for optimal presentation of the data.

2.8 |. Immunocytochemistry/immunohistochemistry staining

Cells and retinal explants were fixed with 4% PFA (Electron Microscopy Sciences) solution at room temperature for 15 min. After fixation, samples were washed with phosphate-buffered saline (PBS) and blocked with the blocking buffer: PBS containing 1% BSA (MilliporeSigma), 0.1% Triton X-100 (MilliporeSigma), and 0.1% Tween20 (MilliporeSigma) for 30 min at 37°C. For 5mc and 5hmc staining in tissues, additional steps were carried out before blocking where tissues were incubated with 0.1% TritonX-100 for 10 min followed by denaturing sections for 30 min with freshly made 2 N hydrochloric (HCl) acid in 1x PBS in 37°C. After this, samples were neutralized using 0.1 M Tris-HCl (pH 8.3) for 10 min. Samples were incubated with primary antibodies (Table S2) at a dilution of 1:100 in blocking buffer at 4°C overnight. The following day, the samples were washed and incubated using respective secondary antibodies (Table S2) at a dilution of 1:500 in blocking buffer for 1 h at 37°C. Finally, the samples were prepares using mounting medium containing DAPI (abcam) and sealed with coverslips. Phase contrast and fluorescent images were obtained using a Leica DMi8 fluorescent microscope. Primary antibody controls were maintained during image acquisition. Neurite images were acquired at day 5 in vitro and day 7 in explants following ES. The number and the length of neurites in all experiments were quantified and analyzed using LasX software and the data graphically represented using GraphPad Prism 5.0.

2.9 |. Quantitative RT-PCR (qPCR)

Cultured cells were washed with PBS, and total RNA was collected using Quick RNA Micro Prep Kit 11–328M (Zymo Research). The quality and concentration of RNA was analyzed using a NanoDrop ND-1000 (Thermo Fisher Scientific). RNA was reverse-transcribed to complementary DNA (cDNA) using Takara Prime Script RT Master Mix RR036A (Takara) using Applied Biophysics 2720 thermal cycler (Life Technologies). Finally, qPCR was done using Power SYBR Green Master Mix (Thermo Fisher Scientific). All primers were purchased from Integrated DNA Technologies (Table S3). A minimum of three biological replicates per treatment group were run with two technical replicates.

2.10 |. Statistical analysis

All data in this study were presented as mean ± standard error of the mean for at least three individual tests. Statistics were analyzed by using GraphPad Prism 5.0. Statistical significance was calculated using the Student t test or one-way analysis of variance as indicated, and a value of p < 0.05 was considered statistically significant. Asterisk denotes * as p < 0.05, ** as p < 0.01, *** as p < 0.001, for all statistical results.

3 |. RESULTS

3.1 |. NGF promotes neurite outgrowth and TET1 expression in PC12 cells

To explore the epigenetic regulation, particularly the involvement of TET expression, in neurite growth capacity of neurons, we adopted PC12 cell model system, which has been widely used to study signaling events regulating neuronal differentiation and neurite outgrowth. PC12 cells respond to NGF by undergoing neural differentiation and neurite extension. To identify the TETs that are critically involved in mediating neurite outgrowth, we treated PC12 with increasing NGF levels (0, 5, and 100 ng/mL), respectively. TET levels were assessed quantitatively in PC12 cells using qPCR at 24 h after NGF treatment, and PC12 neurite outgrowth were quantified 5 days after NGF treatment. We showed that NGF induced neurite outgrowth in a dose dependent manner (Figure 1A–C). Accordingly, qPCR data revealed a dose dependent increase of TET1 (Figure 1D), but not TET2 or TET3, following NGF treatment. The data suggest that TET1 expression correlates well with the neurite outgrowth in PC12 cells.

FIGURE 1.

Nerve growth factor induces PC12 differentiation and TET1 expression. (A) Bright field images of PC-12 cells exposed to increasing concentrations of NGF (0, 5, and 100 ng/mL) exhibited a dose dependent increase in differentiation as indicated by significantly increased neurite outgrowth quantified by (B) neurite length and (C) neurite numbers per field. Images were acquired 5 days post NGF induction of differentiation. (D) Results of qPCR assessments of the levels of TET1, TET2, and TET3 in PC-12 cells exposed to increasing concentrations of NGF. A significant dose dependent increase in TET1 mRNA was observed in PC12 cells. RNA samples were collected 24 h post NGF treatment for qPCR analysis. Values are means ± SEM of indicated number of samples (n). *p ≤ 0.05; ***p ≤ 0.001. mRNA, messenger RNA; NGF, nerve growth factor.

3.2 |. ES facilitates neurite outgrowth and TET1/5hmc levels in PC12 cells

ES has been previously reported to mediate epigenetic factors and is a proven neuronal growth stimulator.^12,15 We thus explored the relation between TET expression and neurite growth in the PC12 cell model system. To avoid the ceiling effect, PC12 cells were subjected to a suboptimal level of NGF (5 ng/mL) and 20 min microcurrent ES. The levels of TET expression were assessed quantitatively by qPCR at 24 h after NGF and ES treatment, and neurite outgrowth was assessed after 5 days of incubation when PC12 cells usually reach the peak of neurite extension. We found that PC12 cells subjected to ES and a low level NGF stimulation showed increased neurite length and numbers compared to non-ES treated controls (Figure 2A–C). The physiognomies of increase in neurite lengths and numbers in ES-treated cultures were similar to that seen in the 100 ng/mL NGF-treated group (Figure S1). No cytotoxic effect was observed in ES-treated group as confirmed by LDH estimation kit and Live and Dead Cell Kit (Figure S2). ES mediated neurite outgrowth was mitigated when cells were pretreated with TETi76 diethyl ester, a pan-TET inhibitor (Figure 2D–F), indicating that the neurite outgrowth is mediated by TETs (DNA demethylation). Results of qPCR in 24 h after ES revealed increased mRNA levels of growth associated protein 43 (GAP43), which encodes a protein highly enriched in growth cones and a positive indicator of growing or regenerating axons, compared to that in non-ES-treated PC12 cells (Figure 2G). Importantly, increased levels of TET1 mRNA expression were again showed by qPCR in ES-treated PC12 cells, corresponding to the increased neurite outgrowth, compared to non-ES controls (Figure 2H). Furthermore, immunostaining of PC12 cells after 24 h of ES confirmed a significant increase in 5hmc expression in their nucleus, supporting an enhanced TET1 activity (Figure 2I). Thus, the results further support correlated increases of TET1 and 5hmc expression with PC12 cell neurite outgrowth following ES-treatment, supporting a TET1-mediated epigenetic event that catalyzes neurite outgrowth.

FIGURE 2.

Electrical stimulation increased neurite outgrowth and DNA demethylation in PC-12 cells. (A–C) ES increased neurite outgrowth in PC12 cells compared to control unstimulated cells. PC12 cells incubated with a low level of NGF (5 ng/mL) (panels 1 and 2) and received 20 min ES at 24 h post NGF induction. (D–F) ES increased neurite outgrowth in PC12 cells compared to control unstimulated cells. PC12 cells incubated with a low level of NGF (5 ng/mL) (panels 1 and 2) and received 20 min ES at 24 h post NGF induction. However, pre-incubating PC12 cells with TETi76 diethyl ester (a pan TET inhibitor) at 12.5 μM (for 16 h) before ES (panel 3), blocked the ES induced neurite outgrowth. Result of qPCR assessing mRNA levels of GAP43 (G) and TET1 (H) expression in Non-ES- and ES-treated cells. (I) Immunocytochemistry based analysis of 5hmc (green) expression, an intermediate product of DNA demethylation process, reveals increased expression of the substrate following ES. The expression was localized in the nucleus (blue). Values are means ± SEM. **p ≤ 0.01; *p ≤ 0.05 by the Student t test. mRNA, messenger RNA; NGF, nerve growth factor.

3.3 |. ES reduced heterochromatin configuration

To explored if ES-mediated TET1 upregulation unwinds the histone-DNA complex following demethylation distancing methylated histones, and hence promoting DNA transcription, we performed chromatin pattern analysis using H3K9me3 (trimethylation of lysine 9 of histone H3) as a marker of chromatin condensation.²⁸ H3K9me3 is a histone modification that frequently occurs alongside DNA methylation and presents a condensed and silenced chromatin configuration (Figure 3A). PC12 cells without ES treatment showed condensed H3K9me3 expression and localization in the nuclei throughout the period (Figure 3B). However, ES treated cells started with a condensed H3K9me3 expression but showed methylated-histone regions moved apart from each other and localized preferably to nuclear periphery at 24 h post-ES (Figure 3C), suggesting ES-induced anti-heterochromatin configuration. Thus, ES-induced TET1 expression is associated with a relaxed heterochromatin configuration that may support gene translation to enable neurite growth-related genes.

FIGURE 3.

Electrical stimulation reduced heterochromatin configuration formation. (A) Graphical illustration (Biorender was used to create the illustration) showing the histone distancing following increased TET1 and DNA demethylation, thereby promoting translational activity. Immunolabeling of H3K9me3 (green) used as a marker to reveal the distribution of histone-DNA complex in the nuclei. Note PC12 cells treated with ES after (B) 20 min (panels 1 and 2) did not show any significant changes in H3K9me3 localization. However, PC12 cells treated with ES after (C) 24 h culturing (panels 1 and 3) showed periphery and distanced localized H3K9me3 with a distinctive (panels 3 and 4) translational pattern compared to non-ES-treated cells. ES condition: 75 μAmp-biphasic/ramp/50 ms for 20 min-once. ES, electrical stimulation.

3.4 |. Correlation between the losses of TET1 expression and axon growth capacity in developing RGCs

Mouse RGCs undergo exponential growth or axon elongation from E11 to E16, while they switch to a slow mode of axon sprouting after birth (P0), and the process ceases after maturation or in adulthood. This phenomenon is known to be regulated by an intrinsic program of neurons and offers a unique opportunity for investigating the gene expression changes in correlation with axonal growth potential.^29,30 Here we performed this correlative study to understand the change in levels of expression of DNA demethylases (TETs) and its dynamic pattern during development. TETs (TET1, TET2, and TET3) are 5 methylcytosine (5mC) dioxygenases responsible for catalyzing the conversion of 5mC to 5hmC (an intermediate during DNA demethylation) thereby promoting open chromatin structure favoring increased transcription (Figure 4A). Primary RGCs were isolated from dissected mouse retinas and quantitative RT-PCR (qPCR) was carried out using these cells for analyzing the epigenetic pattern in different age group of mice. qPCR results using mouse RGCs purified from E-14, P-0, and 3-month-old animals revealed a significant downregulation of all three DNA demethylases, TET1–TET3, by 3 months old (Figure 4B). Moreover, double-immunolabeling of 5mc (DNA methylation substrate) and 5hmc (DNA demethylation intermediate) in similar age group retinas showed prominent expression in the ganglion cell layer (GCL) (Figure 4C), in line with the down-regulations of TETs. This data points toward a dynamic epigenetic axis during RGC development that may act a mechanism leading to reduced regenerative potential of the ON. Diminished 5hmc expression concomitant with reduced TET levels indicates suppressed catalytic activity of the TET demethylase family after maturation or during aging.

FIGURE 4.

Developmental downregulation of TETs in RGCs. (A) Graphical representation of DNA demethylation mechanism and its effect on chromatin structure and translational activity. *thymine-DNA glycosylase (TDG)-mediated base excision repair (BER) (Biorender was used to create the illustration). (B) Quantification of TET1, TET2, and TET3 mRNA with qPCR in the developing mouse RGCs. Note the significantly decreased mRNA levels of all TET(s) with age. Retinas from 4 mice were pooled into two groups (represented as two data points) for the E-14 age group. (C) Images of retinal sections of E-16, P-0 and adult mice (3 months) double-immunolabeled with primary antibodies against 5mc (red) and 5hmc (green); retinal sections were counterstained with a nuclear marker DAPI (blue). Values are means ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 by the Student t test. GCL, ganglion cell layer; INL, inner nuclear layer; mRNA, messenger RNA.

3.5 |. ES promotes neurite growth in mouse and human retinal explants

To verify that ES also promotes the neurite growth in primary RGCs, adult (6 weeks old) mouse eyes were dissected, and the retinal explant was cultured on trans-well membranes. The retinal explants were subjected to ES as described above for 20 min and then incubated for 7 days. The tissues were fixed and immunolabeled for an RGC marker βIII-tubulin (Figure 5A). Quantification results showed that retinal explants subjected to ES exhibited significantly enhanced neurite outgrowth, including increased numbers and average neurite lengths, compared to non-ES controls (Figure 5B,C). A similar experiment was conducted under identical conditions using human retinal explants taken from cadaveric human eyes. ES treatment also induced robust increases of neurite numbers and lengths in human retinas (Figure 6A–C). Increased TET1 expression in ES treated explants were again detected (Figure 6D). Therefore, ES promoted significant neurite outgrowth in the mouse and human retinas that are associated with increased expression of TET1. The results suggest ES as a promising strategy for promoting ON growth or regeneration.

FIGURE 5.

Electrical stimulation increased neurite outgrowth in mouse retinal explant cultures. (A) Immunodetection of β-III tubulin-labeled neurites (arrowhead) in representative adult mouse retinal explant cultures with and without ES. (B, C) Quantification using LASX software reveals significantly longer (B) and increased number of neurites in cultures subjected to ES compared to unstimulated cultures. After 24 h in culture in trans-well membranes, the tissues were subjected to ES. The images were presented in grayscale (panels 1 and 2) for optimal and clear presentation and analysis. Values are mean ± SEM. *p < 0.05 by the Student t test. ES, electrical stimulation.

FIGURE 6.

Electrical stimulation increased neurite outgrowth in human retinal explant cultures. (A) Immunodetection of β-III tubulin (to label neurites) in adult human retinal explant cultures followed by quantification of neurite outgrowth using LASX software. Significantly (B) longer (arrowheads) and (C) a greater number of neurites in retinal explant cultures exposed to ES than in unstimulated cultures was noted. Human and mouse retinal explants underwent identical culture and experimental conditions. (D) Results of qPCR quantification of TET1 exhibited a significant increase in TET1 mRNA in the retinal explants undergone ES versus non-ES controls. The images were presented in grayscale (panels 1 and 2) for optimal and clear presentation and analysis. Values are mean ± SEM of the indicated n. *p < 0.05; **p < 0.01; ***p < 0.001 by the Student t test. ES, electrical stimulation; mRNA, messenger RNA.

3.6 |. ES improves RGC function in ONC model

The effect of ES, especially the functional benefit, was further tested in an in vivo ONC model. ONC leads to axonal degeneration, leading to the death of RGCs and irreversible vision loss. This technique provides an acute and reproducible model of RGC degeneration for the evaluation of novel neuroprotective therapies. Mice after ONC were subjected to ES. Non-ES controls underwent similar conditions without applying the electricity (Figure 7A). Thy1-YFP transgenic mice were subjected to ES every other day starting immediately following ONC. Four weeks after ONC, mice were sacrificed. Long nerve fibers (white arrows) were observed in the retinal nerve fiber layer (RNFL) of ES-treated mice; some could be seen to originate from Thy1 expressing RGCs; whereas, nerve fibers were not seen in the RNFL of retinal sections of the sham group (Non-ES) (Figure 7B). Quantification of fluorescence intensity in Thy1-YFP expressing RGCs confirmed significantly higher number of RGCs bearing neurite outgrowth in ES group retinal sections as compared to non-ES/sham retinal sections following ONC (Figure 7C). To verify that ES modulates DNA demethylation in the ONC model, retinal sections were immunostained for 5hmc. Increased number of Thy1-YFP positive RGCs expressed 5hmc following ES in ONC mice (Figure 7D,E). Conversely, number of Thy1-YFP positive cells expressing 5hmc remained low in non-ES treated group. The functional aspect of the RGCs were tested by pSTR of electroretinogram in mice.³¹ Results of pSTR revealed significantly reduced RGC responses in ONC eyes compared to contralateral non-injured eyes at 4 weeks post crush. Remarkably, the mouse eyes that received ES treatment following ONC exhibited improved RGC function as indicated by significantly higher b-wave amplitude compared to ONC eyes receiving no ES (Figure 7F). These in vivo observations support that ES can rescue RGC following trauma and DNA demethylation is critically involved in RGC axonal survival and functionality after injury.

FIGURE 7.

Electrical stimulation alleviates RGC damage and functional deterioration in ONC model. (A) Graphical abstract of the in vivo experimental plan (Biorender was used to create the illustration). (B) Representative images of immunohistochemistry of Thy1-YFP mouse retinal sections showing YFP+ RGCs (green; cells in the innermost layer) bearing increased nerve fibers in the ES group (arrowheads). (C) Quantification of YFP+ RGCs exhibiting axonal growth in ES and non-ES mice group retinal sections. ***p < 0.001 by Student t test. (D–E) Retinal sections from non-ES and ES-treated Thy1-YFP transgenic mice immunostained for 5hmc. Number of cells co-expressing Thy1-YFP and 5hmc in the ES mice group were higher, but much less seen in non-ES mouse retinas. (F) ES improves ERG responses in ONC mice. Following ONC, the pSTR values dropped significantly compared to noninjured control eye (Cnt); ES-treatment improved the ERG pSTR amplitude. Values are mean ± SEM. *p < 0.05; **p < 0.01 by the Student t test. ES, electrical stimulation; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells.

4 |. DISCUSSION

This study presents data supporting the neuroprotective and regenerative potentials of ES in neurons and RGCs in vitro and in vivo. Additionally, this effect of electric current correlates with its augmentation of TET1 expression. The ERG and immunohistochemistry data in ONC model support functional rescue of RGCs after injury. These outcomes suggest that microcurrent ES present a promising technique for alleviating ocular neurodegeneration, which may offer a whole new dimension of translational therapeutic strategy that can act as a supplemental treatment regime.

Several studies have reported the neuroprotective effect of ES in various ocular diseases.^20,32 These protective effects are not limited to one subtytpe of cell in the retina.³² ES is known to present favorable anti-pathological effect in different models by blocking apoptosis and also promote cellular differentiation.^33,34 However, the upstream molecular players dictating these cellular pathways remain unknown. Our study suggests that ES alters epigenetic modification namely, DNA demethylation or TET expression. DNA demethylation in retina is vital for development,^3,4 and TET knockout mice exhbiit oligodendrocyte differentiation defects and a reduced capability for axon remyelination following injury.^4,5 In ocular injury models of adult mice, DNA methylation reportedly upregulated, resulting in constricted DNA-histone configuration that limits vital gene translation.⁴ Meanwhile, studies have shown that DNA demethylation is effective in axonal regenration following ONC in mice models.^4,35 Vision restoration induced by the pro-regeneration genes, Oct4, Sox2, and Klf4 (OSK) and RGC survival and axon regeneration mediated by OSK and Stat3 upregulation are TET1/2 dependent.⁴ TET1 is required for Pten deletion-induced axon regeneration of retinal RGCs in ONC in vivo models.³⁵ However, as per our knowledge, these two are the only known prominenet studies that report the role of DNA demethylase, TETs, in ON repair. Therefore, building on this knowledge, our study validates that ES mediates axonal growth is dependent on TET1 catalysed DNA demethylation in neurons.

Though there are not many proteomics data establishing the connection between DNA demethylation and RGC regeneration, DNA demethylation is known to influence many established molecular protein based pathways responsible for RGC regeneration.^3,36 Epigenetics, especially DNA demethylation has the potential to dictate the levels of vital downstream molecules, including GAP43³⁷ and Tubb3,³⁸ which are prominently expressed in growth cones of regenerating axons. GAP43 markedly upregulated in electrically stimulated samples in our study as well, an observation reported previously.³⁹ This phenomenon observed in the current study may be the result of increased GAP43 translation owing to electric stimulation induced unwinding of the histone-DNA complex. Deletion of PTEN, SOCS3 and IL22 instigates mTOR and STAT3 pathway activation, pathways known to protect RGCs and mediated via epigenetics.^40–42 Though these are some promising indicators, further proteomic based studies are required to establish the role of epigenentics in RGCs survival and repair. ES mediated increased βIII staining owing to increased neurite outgrowth in this study indicates amplication of pro-RGCs generative genes and protein expression. Therefore, further investigation is required to unravel the underlying mechansims participating in ES based RGCs survival and growth.

Microcurrent electric current is an emerging treatment modality tested in clinical setting for vision enhancement.^43,44 Standardization of optimal electric current intensity, waveform, duration and frequency is required to provide the best treatment outcomes. Earlier reports propose higher intensity microcurrents for longer time duration and using a more invasive transcorneal delivery approach, which can be a hurdle. Therefore, it is imperative that we test alternative parametes that can assure swift and effiecient clinical testing and approval. Previous studies from our lab have established that RAMP waveform for shorter duration administered transpalpebral alleviates photoreceptor damage and also reduces microglial activation.^19,20 In the current study, we report enhanced neuronal differentaition and RGC growth under similar conditions. Hence these combined benefits offered by ES promises a novel treatment strategy to address ocular diseases.

A requirement for effective RGC functionality is a successful integration of RGC dendrites to the retinal layers and the axons directioned towards the ON head.⁴⁵ Though the ES based axonal regeneration in human ex vivo explants seemed limited, we should keep in mind that these are retinas from old-aged individuals with limited regenerative capacity. Therefore, even minimal neurite outgrowth in these tissues is a promising outcome. Finally, most of the ocular diseases including glaucoma that effect the RGCs are exacerbated by co-exisitng conditions such as inflammation.⁴⁶ Reports from our lab and earlier studies have shown that ES attenuated the expression of proinflammatory cytokines namely IFN-γ, TNF-α, and IL-1α, thereby adding an extra protective axis to its neuroprotective effects.¹⁹ Therefore, ES presents itself as a multifaceted therapeutic tool to address ocular pathologies.

In conclusion, our data cumulatively suggest that ES has the potential to regulate DNA demethylation, which is connected to its potential of promoting neuronal differentiation in PC12 cells and RGC axonal growth. Further studies are required to understand the molecular pathways that are modulated by ES in association with its ability to alter DNA methylation. Moreover, the established safety profile of low intensity current in different phases of testing a provides a basis for further investigation of noninvasive ES as a neuroprotective and regenerative treatment option to address ocular diseases.

Abbreviations:

ES

electrical stimulation

RGCs

retinal ganglion cells

TET

ten-eleven translocation

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.