Electrical stimulation enhances mitochondrial trafficking as a neuroprotective mechanism against chemotherapy-induced peripheral neuropathy

Summary Electrical stimulation (ESTIM) has shown to be an effective symptomatic treatment to treat pain associated with peripheral nerve damage. However, the neuroprotective mechanism of ESTIM on peripheral neuropathies is still unknown. In this study, we identified that ESTIM has the ability to enhance mitochondrial trafficking as a neuroprotective mechanism against chemotherapy-induced peripheral neuropathies (CIPNs). CIPN is a debilitating and painful sequalae of anti-cancer chemotherapy treatment which results in degeneration of peripheral nerves. Mitochondrial dynamics were analyzed within axons in response to two different antineoplastic mechanisms by chemotherapy drug treatments paclitaxel and oxaliplatin in vitro. Mitochondrial trafficking response to chemotherapy drug treatment was observed to decrease in conjunction with degeneration of distal axons. Using low-frequency ESTIM, we observed enhanced mitochondrial trafficking to be a neuroprotective mechanism against CIPN. This study confirms ESTIM enhances regeneration of peripheral nerves by increased mitochondrial trafficking.


Highlights
Pulsed electrical Stimulation has the ability to increase axonal growth rates at low frequencies of 10 Hz, 100 Hz, and 1,000 Hz Electrical stimulation has neuroprotective abilities against common chemotherapy-induced peripheral neuropathies (CIPNs) Pulsed electrical stimulation has the ability to increase anterograde mitochondrial trafficking rates at low frequencies of 10 Hz, 100 Hz, and 1,000 Hz A potential mechanism behind electrical stimulation-mediated neuroprotection is increased mitochondrial trafficking

INTRODUCTION
The use of electrical stimulation (ESTIM) on peripheral nerves has been well studied since 1967, when Wall and Sweet reported on the use of ESTIM for pain relief for the first time. 1For the past 50 years, neuromodulation has been implemented in many ways to reduce pain, or as a treatment method for neurological diseases and conditions. 2 Although peripheral nerves have significant regenerative capabilities compared to other cell types, functional recovery is proven to be poor.ESTIM is currently available to the public as a treatment strategy to mitigate pain seen in the form of a transcutaneous electrical nerve stimulator (TENS). 35][6] Many researchers believe that the use of ESTIM on peripheral nerve injuries may be a key therapy for peripheral nerve regeneration in clinical settings. 7Pulsed ESTIM has shown to be an effective treatment strategy for peripheral injury conditions. 8,9Previous studies have shown that using a low-frequency (>100 Hz) pulsed biphasic stimulation for 1 h per day is able to increase sensory nerve regeneration and myelination.Low-frequency stimulation is also considered to be optimal conditions for nerve growth and protection; 7,[10][11][12] however, it is important to identify a larger range of frequencies to fully understand the effect of ESTIM against chemotherapy-induced peripheral neuropathy (CIPN) by paclitaxel (PTX) and oxaliplatin (L-OHP). 7,13,14Although ESTIM has been a well-researched phenomenon, the underlying mechanism regarding peripheral nerve regeneration is poorly understood.
Mitochondrial population and trafficking are significant indicators of neuronal health. 15Low population and trafficking of mitochondria along the axons can be directly related to low energy production, which in turn can lead to peripheral nerve degeneration and cell death. 16nergy demands within the neuron change dependently on location, circumstance, and injury. 17For example, the growth cone and synapse require significant energy resources in order to deliver signals to target locations. 18Due to this high, dynamic energy demand, mitochondria are needed in different locations as well as in different densities to supply energy in the form of ATP on demand. 19Mitochondrial trafficking and dynamic responses help maintain homeostasis regarding energy demands.Neuronal activity has been found to affect mitochondrial trafficking responses, where increased signaling and neuronal activity increase mitochondrial trafficking in localized regions of the neuron. 20The dynamic nature of energy distribution by mitochondrial trafficking is an important mechanism to understand, due to increased energy demands in a disease state such as CIPN. 21,22The effect of specific drugs and treatment methods on mitochondrial transport can help provide an indication on the targeting mechanism for such treatment strategies.Using this information, better methods can be developed to alter subcellular trafficking for neuroprotection.Using imaging techniques, it is possible to determine the trafficking response and communication between transport mechanisms.
CIPN is a debilitating health condition associated with common chemotherapy drugs. 23CIPN is a painful side effect of chemotherapy treatment which affects 67% of all cancer patients undergoing chemotherapy treatment. 24Chemotherapy is an effective treatment method targeting the cancer cell division process, to eliminate cancer from the body.However, there are many adverse side effects as a result of chemotherapy treatment, including peripheral neuropathy.Currently, there are no treatment methods for peripheral neuropathy other than symptom mitigation. 25CIPN is the most common dose-limiting factor during chemotherapy treatment, oftentimes leading to suboptimal conditions to fight cancer, and even termination of treatment in extreme cases.Using this knowledge ESTIM is shown to be an effective treatment option against common chemotherapy drugs, L-OHP and PTX.L-OHP and PTX were chosen due to their common application in chemotherapy treatment which results in CIPN, as well as to compare against different targeting mechanisms.
PTX is known as an effective agent in disrupting the cell division cycle of cancer cells by targeting the microtubules associated with mitosis. 26PTX is primarily used against breast, lung, and ovarian cancer types. 27Due to the microtubule targeting effect of PTX, it can be assumed that PTX is directly related to axonal trafficking.The effect of ESTIM on PTX-induced peripheral neuropathy (PIPN) was studied based on our recent study identifying fluocinolone acetonide (FA) as a neuroprotective agent, which enhances mitochondrial trafficking leading to neuroprotection against common CIPNs. 28To fully understand the mechanism of neuroprotection by ESTIM, another drug with different targeting mechanism is needed for comparison.L-OHP is a third-generation platinum-based antineoplastic chemotherapy drug primarily used to target colorectal and stage III colon cancer. 29L-OHP targets the DNA replication cycle by binding to DNA strands and disrupting the transcription process within cancer cells. 30DNA damage associated with L-OHP treatment is crucial to anti-cancer treatment as it can disrupt tumor development.CIPN is the leading side effect associated with L-OHP treatment, which can cause major discomfort and pain leading to dosage reductions for patients. 31The specific mechanisms of toxicity in CIPN for L-OHP are still unknown; however, it is speculated that transporter-mediated uptake of L-OHP triggers pathophysiological changes seen as disrupted cell signaling cascades, mitochondrial disfunction, and oxidative stress in dorsal root ganglion neurons (DRGs).L-OHP also has been noted to have a higher toxicity effect on mitochondria within targeted cells. 32,33Using this information, we seek to determine if ESTIM is capable of protecting mitochondrial transport even when directly targeted by L-OHP.Therefore, L-OHP and PTX were chosen to determine if ESTIM was effective at enhancing mitochondrial trafficking and subsequent neuroprotection.The two different mechanisms of action for these drugs are the most commonly used types of chemo drugs, and it is important to understand how ESTIM affects both mechanisms.In order to analyze the effect of ESTIM-mediated neuroprotection against PTX-and L-OHP-induced peripheral neuropathy, axon length responses were measured with varying pulsed ESTIM frequencies (10 Hz-1 MHz).DRGs were similarly stimulated using a compartmentalized cell culture chamber to determine the effect of the frequency range on mitochondrial trafficking.Frequencies which showed positive axon length and mitochondrial responses were used in conjunction with PTX and L-OHP to determine the neuroprotective effect against CIPN.This is the first time that ESTIM is shown to have the ability to enhance mitochondrial trafficking in axons that guarantees neuroprotection against CIPN.

Experimental setup
Figure 1 illustrates the experimental schematic for the outlined ESTIM experiments.DRGs were isolated and processed from E-15 pregnant rats and cultured on a custom 12-well compartmentalized culture plate seen in Figure 1.Chemotherapy drugs and fluorescent dyes were treated in culture chambers and incubated for 1 h prior to ESTIM.ESTIM was performed by sending biphasic square pulse wave signals from a Siglent waveform generator to platinum electrodes attached to the lid of the culture device.Imaging using a Leica-DMi8 fluorescent microscope was performed before and after 1 h of stimulation to determine axon length or mitochondria trafficking response.

Global stimulation frequency response
To determine the effect of each frequency on axon length, DRGs were seated in a non-compartmentalized sterile 24-well glass-bottom cell culture plate shown in Figure 2A.DRGs were cultured and allowed to grow for 24 h prior to stimulation. 1 h stimulation at 10 Hz, 100 Hz, 1 kHz, 100 kHz, and 1 MHz was induced to the cells once per day for 3 days as shown in Figures 2B and 2C.Cells were treated with Calcien acetoxymethyl ester (AM) and imaged before and after stimulation using a fluorescent microscope.Fluorescent images were processed using Fiji-ImageJ to measure axon length at each corresponding frequency and timestamp.As seen in Figures 2B and 2C, the frequency response was characterized, where low frequencies (>1 kHz) enhanced axon growth, whereas high frequencies (<1 kHz) had a detrimental effect on axon length.Over the three-day stimulation period, 10 Hz accelerated growth by 9.71% G 0.26%, 100 Hz accelerated growth by 12.28% G 0.33%, and 1 kHz accelerated growth by 0.07% G 0.27%, whereas 100 kHz inhibited growth by 24.43% G 0.21% and 1 MHz inhibited growth by 49.03% G 0.27%.Based on these values, optimal frequencies were chosen when preforming the experiment alongside chemotherapy drugs to characterize the neuroprotective effect of each frequency.Seen in Figure 2B, representative figures for 0, 24, and 48 h when exposed to the 5 different frequencies are shown.Graphical representation of axon length as a comparison to the control can be seen from Figure 2C.After 24 and 48 h, 100 Hz had the highest growth rate.These results are found to be consistent with current research, showing low-frequency stimulation to have the largest positive impact on axon growth.
Fluorescent imaging of mitochondria was performed for each frequency to characterize the trafficking response to the full frequency range (10 Hz-1 MHz).In order to properly measure the frequency response on mitochondrial trafficking, DRGs were seated into the polydimethylsiloxane (PDMS)-based microchannel device and allowed to grow for 3-5 days in order to ensure proper growth through the microchannels.MitoView Green dye was then added to the cell body chamber and incubated in correspondence with the manufacturer's protocol.Fluorescent imaging of mitochondria before and after stimulation at each frequency can be seen in Figure 2F.The specific timestamps in Figure 2C were chosen to measure the direct impact of each frequency on mitochondrial count after 1 h stimulation.Variables, such as dye degradation, media changes, and light exposure, had the potential to skew data if recorded over an extended period of time; therefore, mitochondrial images were collected directly before and after 1 h stimulation at each frequency.A microfluidic compartmentalized cell culture system was used to properly stimulate and identify mitochondrial movement within the axons.Using a parallel array of microchannels, DRGs were compartmentalized in order to fully isolate the cell bodies from the axons.By doing this, axons are aligned perfectly within the microchannels, enabling us to image and count mitochondrial population at each timestamp.MitoView Green dye was placed into the cell body chamber of the compartmentalized system, while neurobasal media were added in higher volume to the axonal chamber, inducing hydrostatic pressure to prevent excess dye diffusion.Cell body dye administration further helped show anterograde movement of mitochondria to focus on the change in energy demand at the site of injury during CIPN.Using these data, we were able to understand how mitochondria were affected by the initial stimulation.After 1 h stimulation, mitochondria population increased by 62.79% G 0.43% for 10 Hz, 62.59% G 0.71% for 100 Hz, and 15.96% G 0.16% for 1 kHz but decreased by 38.90% G 0.19% for 100 kHz and 74.28% G 0.04% for 1 MHz.All values were normalized according to the 0-h control count to provide a proper increase/decrease ratio.Values above ''1.0''represent an increase in mitochondrial number, whereas values below ''1.0'' represent a decrease.The trends seen in the results for mitochondrial population match those with axon length measurements leading us to believe ESTIM directly modulates mitochondrial trafficking and other subcellular axonal trafficking mechanics to increase axonal growth.

Global ESTIM as a neuroprotective method against CIPN
The same global stimulation experimental setup was used to identify the neuroprotective effect of ESTIM against CIPN by PTX and L-OHP shown in Figures 3 and 4, respectively.Optimal concentrations for PTX and L-OHP with in vivo experimentation range from 5 to 20 mg/kg and 5-25 mg/kg, respectively. 34,35Based on in vivo settings, in vitro concentrations were chosen which highlighted proper dying-back neuropathy.PTX and L-OHP were tested using concentrations 2 mM, 5 mM, and 10 mM, where axon length and mitochondrial response were characterized.For each concentration, neurons were stimulated using 10 Hz, 100 Hz, and 1 kHz.The optimal concentration of PTX and L-OHP was determined to be 5 mM, where PTX and L-OHP showed degradation of axons without inducing sudden cell death. 22,36,37Based on the toxicity response of PTX and L-OHP, concentrations of 5 mM are used for additional results.Axon length results showed that PTX and L-OHP at 5 mM had an average degradation of 67.85% and 65.97% after 3-day exposure compared to control, respectively.For PTX, ESTIM was found to inhibit degradation, where 10 Hz showed 15.55%, 100 Hz, 38.57%, and 1 kHz, 39.75% degradation, showing a decrease in PTX degradability where 10 Hz had the highest protection.Alternatively, ESTIM was found to be neuroprotective against L-OHP treatment where 10 Hz showed 48.12%, 100 Hz, 53.74%, and 1 kHz, 54.95% degradation.Although PTX and L-OHP had different targeting mechanisms, ESTIM was found to have a neuroprotective effect on both the drugs.This result leads us to conclude that the underlying mechanism of neuroprotection by ESTIM could be similar if not the same.Processed DRGs plated into the cell body chamber of the compartmentalized chamber system.Cells are allowed to grow for 3-5 days until they grow through the channels to the axonal compartment, where chemotherapy drugs and dyes are administered.Electrical stimulation was performed for 1 h per day using a constant 3 V power supply to generate a biphasic rectangular pulse lasting 0.5 s every 2 s with a pulse width of 0.2 ms.Imaging of axons was completed in the axonal compartment, while mitochondrial trafficking imaging was performed within the parallel microchannels.Created with BioRender.com.

Focal stimulation as a neuroprotective method against CIPN
Axonal stimulation was used to identify the neuroprotective effect against PTX-and L-OHP-induced CIPN.It was important to understand any differences between global and focal stimulation due to treatment application of ESTIM against CIPN.Focal vs. global ESTIM can provide important data regarding treatment methodologies in a clinical setting.Focal stimulation of DRGs was performed by placing both electrodes in the axonal compartment.In a previous paper, it was determined that the electric field was localized to the axonal compartment when preforming focal stimulation. 38The same parameters and experimental setup were used in order to ensure proper electrical field isolation.Chemotherapy drugs were added in both compartments, mimicking global treatment similar to Figures 2, 3, and 4. ESTIM was performed in the axonal compartment using the same optimized parameters as those of the global stimulation.Focal ESTIM proved to be just as effective as global stimulation for neuroprotection against CIPN seen in Figure 5. Axon length results showed that PTX and L-OHP at 5 mM had an The axonal response after stimulation for 0, 24, and 48 h, for 10Hz, 100Hz, 1 kHz, 10 hHz, 100 kHz, and 1 MHz showed that low frequency (10 Hz, 100 Hz, and 1 kHz) proved to enhance axon length measurements at each timestamp (B).Mitochondrial imaging was performed in a compartmentalized microfluidic chamber to identify the immediate trafficking response to each frequency (C).Quantification of normalized axon length measurements were used to compare against the initial 0 h length (D).Mitochondria were shown to increase between the 0 h and 1 h stimulation for low frequency stimulation parameters (E).Mitochondria were imaged before and after 1 h stimulation to identify the trafficking enhancement by ESTIM (F).It was proven that 100 Hz stimulation enhanced mitochondrial trafficking the most compared to control samples (E).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test (E), Scale bar is 100 mm unless otherwise stated.
average degradation of 78.69% and 51.66% compared to control, respectively, seen in Figures 5B, 5C, 5F, and 5G.For PTX-treated samples, ESTIM was found to have a neuroprotective effect, where 10 Hz showed only 39.65%, 100 Hz, 27.38%, and 1 kHz, 34.78% degradation, showing a decrease in PIPN effect on DRGs, where 10 Hz had the highest neuroprotection.Alternatively, ESTIM was also found to be neuroprotective against L-OHP treatment where non-stimulated samples showed 51.66% degradation; however, 10 Hz showed 42.58%, 100 Hz, 33.25%, and 1 kHz, 30.48% degradation.Focal stimulation effect in response to PTX and L-OHP can be seen in Figure 5.

Mitochondrial motility
Figure 6 shows representative time-lapse imaging captured during a 30-min stimulation period.Using a mitochondrial staining approach, displacement of motile mitochondria using time-lapse imaging was determined. 28Using MitoView Green dye, mitochondrial displacement was characterized in response to optimal stimulation parameters.DRGs were cultured in a non-compartmentalized glass-bottom culture dish.A non-compartmentalized device was used due to the ease of use, and mitochondrial clarity when stained.Using 100 Hz global ESTIM, displacement of mitochondrial movement was determined in response to PTX and L-OHP treatment.Displacement over the 30-min time period was determined to be 3.76 G 1.55 mm on average for untreated samples and increased to 5.96 G 1.38 mm during 100 Hz stimulation.PTX treatment measured displacement to be reduced to 2.53 G 0.94 mm but increased to 4.43 G 1.48 mm when stimulated at 100 Hz.Similarly, L-OHP treatment showed an average displacement of 2.31 G 0.78 mm, increased to 4.95 G 1.63 mm when stimulated at 100 Hz.All samples were dyed and treated for 1 h prior to time-lapse imaging.Samples were stimulated using the same parameters as those in all other experiments.Based on velocity determination, 100 Hz stimulation was able to accelerate mitochondrial trafficking by 58.36% for untreated, 42.90% for PTX, and 53.36% for L-OHP.Changes in velocity represent significant movement of motile mitochondria in response to the different conditions outlined.Motile mitochondria were analyzed due to their ability to regulate energy demands along the axon. 39Based on the results from Figure 6, neuroprotection by ESTIM-enhanced mitochondrial transport can further be assumed.

DISCUSSION
ESTIM has a great potential as a symptomatic pain management option for chronic pain conditions. 40In this study, we determined the neuroprotective effect of ESTIM against CIPN.Prior to determination, optimal frequencies for stimulation were determined.Optimal parameters were used to analyze the axonal response to ESTIM-mediated neuroprotection.ESTIM was found to increase axon length when stimulated at low frequencies; however, it was inhibited at higher frequencies (>1 kHz).Mitochondrial trafficking response was then characterized using the full range of frequencies, where only low frequencies (<1 kHz) showed enhanced trafficking effect.Using the optimal frequencies, PTX-and L-OHP-treated samples were stimulated to determine ESTIM-enhanced neuroprotection and mitochondrial trafficking against CIPN.
This study focused on axonal regeneration and trafficking response to global ESTIM using a frequency range of 10 Hz, 100 Hz, 1 kHz, 10 kHz, 100 kHz, and 1 MHz.Results from the frequency response showed that, over the three-day stimulation period, 10 Hz accelerated axonal growth by 9.71% G 0.26%, 100 Hz accelerated growth by 12.28% G 0.33%, and 1 kHz accelerated growth 0.07% G 0.27%, whereas 10 kHz inhibited growth by 24.43% G 0.21%, 100 kHz inhibited growth by 24.43% G 0.21%, and 1 MHz inhibited growth by 49.03% G 0.27%.As expected, low-frequency (<1 kHz) stimulation increased growth rates of DRGs.Historically, ESTIM has been reported to increase neural activity within neuron cells when exposed to low-frequency stimulation. 41The increased neural activity induced by low-frequency stimulation within embryonic DRGs showed increases in axonal growth rates significantly. 42Subsequently, mitochondria trafficking was analyzed due to its significance in energy production and overall health of the neuron, as well as its association with neurodegenerative disorders. 43,44Mitochondrial trafficking response to the frequency range was analyzed using fluorescent dyes to take live image of the effect of ESTIM.ESTIM enhanced mitochondrial trafficking by 62.79% G 0.43% for 10 Hz, 62.59% G 0.71% for 100 Hz, and 15.96% G 0.16% for 1 kHz but decreased 33.78% G 0.18% for 10 kHz, 38.90% G 0.19% for 100 kHz, and 74.28% G 0.04% for 1 MHz.Control samples showed mitochondrial population increase of 0.05% G 0.09% which can be attributed to mitochondrial motility under normal conditions.Although no external stimulation was provided, motile mitochondria are still seen to traffic in an anterograde manner shown in Figure 2 due to changing energy demands. 39Due to the increased neural activity induced by ESTIM, we believe total axonal transport was enhanced as shown by the mitochondrial trafficking data.6][47] Enhanced mitochondrial trafficking is likewise heavily associated with increased ATP levels. 48Increased ATP by enhanced mitochondrial trafficking is directly correlated with neuroprotection in peripheral nerve injuries. 28,49The findings for axon length and mitochondrial trafficking correlate significantly, as the most effective increase in trafficking and axon length seemed to be 100 Hz.High-frequency stimulation (>1 kHz) showed a decrease in axon length and mitochondrial trafficking.These findings were expected as high-frequency stimulation is a Figure 3. Electrical stimulation enhances mitochondrial trafficking as a neuroprotective mechanism against PTX Global stimulation setup using 10 Hz, 100 Hz, and 1 kHz (A).(B) Schematic of mitochondrial dynamics during ESTIM.PTX-treated samples were stimulated for 1 h per day using the same parameters as frequency response (C).Quantification of neuroprotection by ESTIM against PTX 5 mM-induced peripheral degeneration (D).(E) Quantification of normalized mitochondrial count as compared to control samples during ESTIM for PTX 5 mM.Quantification of neuroprotection by ESTIM against PTX 2 mMand 10 mM-induced degeneration, respectively (I, J).Mitochondrial imaging was performed using the compartmentalized chamber system where imaging was preformed within microchannels after axons grew through (A).Image analysis of mitochondrial trafficking effect of 1-h ESTIM after PTX treatment using 2 mM, 5 mM, and 10 mM (F-H).Quantification of mitochondrial trafficking effect by ESIM against PTX 2 mM and 10 mM (K, L).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test, Scale bar is 100 mm unless otherwise stated.known phenomenon to induce excitotoxicity in neurons. 50Due to the negative effect of high-frequency stimulation, further experimentation opted to not use these frequencies.The global stimulation results show significant findings regarding mitochondrial trafficking enhancement, where proper application could result in neuroprotection against CIPN.
In this study we show increased mitochondrial trafficking because of low-frequency ESTIM; however, the underlying mechanism behind this observation is still unclear.Future studies are necessary to determine signaling pathways associated with ESTIM-enhanced mitochondrial trafficking.Herein, we believe one potential mechanism that could be attributed to enhanced trafficking observed in this study is increased brain-derived neurotrophic factor (BDNF) expression. 51BDNF is thought to have a direct association with peripheral nerve regeneration, due to its increased expression in injured conditions.BDNF is shown to have rapid upregulation which can be sustained for weeks, strongly suggesting its role in regeneration. 52,53One study suggests that during ESTIM intracellular Ca 2+ is increased, leading to overexpression of BDNF. 54,55Recently, BDNF is believed to modulate mitochondrial trafficking through downstream activation of protein kinase A. 56 Further research is needed to correlate enhanced BDNF expression by ESTIM and enhanced mitochondrial trafficking.
Previously we determined that PTX has the ability to inhibit mitochondrial transport when treated on sensory neurons. 22,28Based on this finding, we sought to determine the effect of ESTIM as a neuroprotective mechanism against two different antineoplastic drugs, PTX and L-OHP.Global stimulation with chemotherapy drug treatment was initially performed to analyze the effect of ESTIM on axon length and mitochondrial trafficking, similar to the frequency response experiment.PTX is a common microtubule targeting anti-cancer drug, which in turn affects the axonal transport mechanisms leading to peripheral degeneration.From our results, 10 Hz stimulation provided the highest degree of neuroprotection against PIPN, significantly reducing the degeneration effects of PTX compared to control sample.Likewise, mitochondrial trafficking was enhanced, where 100 Hz provided the largest change in trafficking after stimulation.Based on ESTIM's ability to enhance mitochondrial trafficking, it was speculated that if we could enhance the axonal trafficking within the cell in response to PTX treatment, it would provide neuroprotection against PIPN.Similarly, L-OHP was treated globally at a concentration of 5 mM based on in vitro toxicity response. 36,37Due to the DNA targeting mechanism of L-OHP treatment, the effects of enhanced axonal transport were expected to be less than those of PTX.L-OHP treatment targets DNA replication cycle within the nucleus, and it is assumed to likewise affect the DNA within mitochondria. 57,58Mitochondrial observation is key in response to L-OHP treatment as mitochondria has recently been associated with signaling responses associated with cell death. 59Although L-OHP targets DNA replication, ESTIM is still shown to have a neuroprotective effect on L-OHP-treated samples.Mitochondrial trafficking was also analyzed for L-OHP-treated samples to further understand how enhanced axonal transport can be used as a neuroprotective mechanism against CIPN.Although L-OHP has a different targeting mechanism than PTX, based on the results it can be seen that low-frequency stimulation still enhanced anterograde mitochondrial trafficking, where 100 Hz showed the highest increase over the stimulation period.It is important to note that mitochondrial trafficking enhancement was surprisingly similar when comparing PTX and L-OHP.Although axon length analysis showed a difference in results for each drug, mitochondrial enhancement was similar between the two, potentially indicating a similar enhancement mechanism by ESTIM.Based on our results, the axonal transport enhancement by ESTIM proved an effective neuroprotection mechanism against CIPN.Further research is needed to determine if ESTIM directly affects mitochondria trafficking as a primary or secondary mechanism.Based on this study, we speculate that the increased neural activity by low-frequency stimulation enhances axonal transport; however, the specific signaling mechanisms are still unknown at this time.Further research is needed to fully understand the signaling pathways behind neuromodulation-enhanced regeneration.Based on the results found, ESTIM has the ability to be an effective method to help patients who are experiencing painful CIPN.
In order to determine the most effective treatment method for ESTIM, axonal stimulation was performed on PTX-and L-OHP-treated samples.It was necessary to understand if there were any major differences between global and axonal stimulation since axonal stimulation is a preferred treatment method in clinical settings.Axonal stimulation allows for isolated electric fields at the region of injury, which in turn can be a safer and more effective treatment option for patients.Axonal stimulation effect against PTX-and L-OHP-induced degeneration was significant due to CIPN being a focal injury model in patients.For PTX-treated samples, 100 Hz stimulation had the highest neuroprotective effect, while for L-OHP-treated samples, 100 Hz and 1 kHz stimulation had equally the highest protection.Based on the results, axonal and global stimulation had similar effectiveness, justifying the ability to use focal stimulation as an effective method for neuroprotection against CIPN.The similarities between global and focal stimulation could be attributed to the electric field distribution within the cell.Previous studies have confirmed that single-cell stimulation can illicit increased neural activity throughout the entire cell in vitro and in vivo. 38,60Axonal stimulation does not have any lesser effect on neuroprotection than global and can be determined as an effective treatment method in clinical settings.
Although mitochondrial trafficking was the governing subcellular organelle that was studied, it may not be the only organelle that is affected by neuromodulation.Further research is needed to determine how other mobile organelles are affected by ESTIM.To help answer the question of enhanced axonal transport as a neuroprotective mechanism, a supplementary lysosome trafficking study was performed as a supplementary study shown in Figure S1.The effect of lysosome trafficking mirrored mitochondrial data, showing that ESTIM enhanced trafficking of lysosomes within DRGs.This supplementary data help back up the claim that ESTIM may enhance axonal trafficking as a neuroprotective mechanism against CIPN.Focal pulsed stimulation on DRGs using the microfluidic compartmentalized cell culture chamber Global drug treatment was administered using PTX and L-OHP with optimal concentrations (A).Axon length measurements were collected and quantified in response to PTX treatment and ESTIM (B, C).Mitochondrial imaging was performed before and after stimulation at each frequency (D, E).Axon length measurements were collected and quantified in response to L-OHP treatment and ESTIM (F, G).Mitochondrial imaging was performed before and after stimulation at each frequency (H, I).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test, Scale bar is 100 mm unless otherwise stated.
Based on the results of this study, mitochondrial enhancement by ESTIM is a proposed mechanism behind ESTIM-mediated neuroprotection against CIPN.Mitochondrial trafficking enhancement shows to be a key aspect of neuroprotection, which has the potential to help many patients suffering from CIPN.Based on this study, non-invasive techniques can be developed to generate electric fields, enhancing mitochondrial trafficking in clinical settings to treat CIPN or other neurodegenerative disorders or diseases.

Limitations of the study
There were several limitations of this study.First, mitochondrial trafficking dye has an inherent diffusion rate, which may influence the trafficking analysis.Hydrostatic pressure and flow induction were performed to reduce this aspect; however, we have no way to ensure total blockage of dye diffusion at this time.Displacement over time data were manually recorded, where time-lapse images showed frame shifting, and axonal degradation in response to chemotherapy drugs, making it difficult to collect accurate velocity data from mitochondrial trafficking.At this time, we are unable to determine the signaling pathways and direct mechanism associated with ESTIM-enhanced mitochondrial transport.We hope to address all these challenges in future studies.

Figure 1 .
Figure1.Experimental schematic E-15 Sprague-Dawley rat embryonic dorsal root ganglion (DRG) isolation and enzymatic disassociation.Processed DRGs plated into the cell body chamber of the compartmentalized chamber system.Cells are allowed to grow for 3-5 days until they grow through the channels to the axonal compartment, where chemotherapy drugs and dyes are administered.Electrical stimulation was performed for 1 h per day using a constant 3 V power supply to generate a biphasic rectangular pulse lasting 0.5 s every 2 s with a pulse width of 0.2 ms.Imaging of axons was completed in the axonal compartment, while mitochondrial trafficking imaging was performed within the parallel microchannels.Created with BioRender.com.

Figure 2 .
Figure 2. Pulsed electrical stimulation enhances axon growth and mitochondrial traffickingSchematic for global DRG stimulation for axon length measurement analysis (A).The axonal response after stimulation for 0, 24, and 48 h, for 10Hz, 100Hz, 1 kHz, 10 hHz, 100 kHz, and 1 MHz showed that low frequency (10 Hz, 100 Hz, and 1 kHz) proved to enhance axon length measurements at each timestamp (B).Mitochondrial imaging was performed in a compartmentalized microfluidic chamber to identify the immediate trafficking response to each frequency (C).Quantification of normalized axon length measurements were used to compare against the initial 0 h length (D).Mitochondria were shown to increase between the 0 h and 1 h stimulation for low frequency stimulation parameters (E).Mitochondria were imaged before and after 1 h stimulation to identify the trafficking enhancement by ESTIM (F).It was proven that 100 Hz stimulation enhanced mitochondrial trafficking the most compared to control samples (E).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test (E), Scale bar is 100 mm unless otherwise stated.

Figure 4 .
Figure 4. Electrical stimulation enhances mitochondrial trafficking as a neuroprotective mechanism against L-OHP L-OHP-treated samples were stimulated for 1 h per day using the same parameters as frequency response (A).Quantification of neuroprotection by ESTIM against L-OHP 5 mM-induced peripheral degeneration (B).(C) Quanification of normalized mitochondrial trafficking effect during ESTIM at L-OHP 5 mM.Quantification of neuroprotection by ESTIM against L-OHP 2 mMand 10 mM-induced degeneration respectively (G, H).Image analysis of mitochondrial trafficking effect of 1-h ESTIM after L-OHP treatment using 2 mM, 5 mM, and 10 mM (D-F).Quantification of mitochondrial trafficking effect by ESIM against L-OHP 2 mM and 10 mM (I, J).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test, Scale bar is 100 mm unless otherwise stated.

Figure 5 .
Figure 5. Focal pulsed stimulation on DRGs using the microfluidic compartmentalized cell culture chamber Global drug treatment was administered using PTX and L-OHP with optimal concentrations (A).Axon length measurements were collected and quantified in response to PTX treatment and ESTIM (B, C).Mitochondrial imaging was performed before and after stimulation at each frequency (D, E).Axon length measurements were collected and quantified in response to L-OHP treatment and ESTIM (F, G).Mitochondrial imaging was performed before and after stimulation at each frequency (H, I).Data are shown by mean G SD. ***p < 0.001, **p < 0.01, *p < 0.05 as determined by two-tailed t test, Scale bar is 100 mm unless otherwise stated.