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Article

Preliminary Study on Safety Assessment of 10 Hz Transcranial Alternating Current Stimulation in Rat Brain

by
Sung Suk Oh
1,
Yoon Bum Lee
2,
Jae Sun Jeon
1,3,
Sang-Hyun An
2 and
Jong-ryul Choi
1,*
1
Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI Hub), Daegu 41061, Korea
2
Preclinical Research Center, Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI Hub), Daegu 41061, Korea
3
School of Biomedical Engineering, Daegu Catholic University (DCU), Gyeongsan 38430, Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(11), 5299; https://doi.org/10.3390/app12115299
Submission received: 18 April 2022 / Revised: 20 May 2022 / Accepted: 20 May 2022 / Published: 24 May 2022
(This article belongs to the Section Biomedical Engineering)
Browse Figures
Versions Notes

Abstract

:
Assessment of the safety of transcranial electrical stimulation devices that contact the scalp and apply electrical stimulations to brain tissues is essential for the prevention of unexpected brain damage caused by electromagnetic fields. In particular, safety studies on transcranial alternating current stimulation (tACS) are needed for active applications to treat brain diseases and for the development of medical devices, because there is a lack of research on the safety of tACS, in contrast to transcranial direct current stimulation. In this study, the safety of tACS with selected parameters, i.e., a stimulation intensity of 1.0 to 2.0 mA, a frequency of 10 Hz, and a treatment time of 20 min, was examined at a preclinical stage using small animals (rats). The results of magnetic resonance imaging and histopathological imaging indicated that the conditions applied in this study provided safe tACS without damaging brain tissues or neuronal components in the acute phase. In addition, the temperature did not increase above 41 °C, which is a temperature limitation for contact-type medical devices, even after 20 min of tACS application.

1. Introduction

Transcranial electrical stimulation is a treatment that delivers weak electrical stimulation to the brain surfaces through electrodes located above the scalp for the spontaneous activation of neuronal cells to repair brain function and relieve symptoms [1,2,3,4,5]. Transcranial electrical stimulation can be classified as transcranial direct current stimulation (tDCS), which delivers a direct current without frequency modulation to the brain; transcranial alternating current stimulation (tACS), which delivers a modulated electrical current at a constant frequency; and transcranial random noise stimulation (tRNS), which delivers a current with randomly changing frequencies and intensities [6,7]. In tDCS, depending on the characteristics of the direct current flowing from the anode to the cathode, brain function is controlled by placing an anode on the head to induce depolarization, which increases the neuronal excitability (anodal tDCS), or by placing a cathode on the head to induce hyperpolarization, which reduces the neural excitability (cathodal tDCS). In contrast, in tACS, which delivers alternating currents without a constant current direction, brain functions are adjusted by causing brain oscillatory activities at a specific frequency. Recently, results of preclinical in vivo studies have been reported to understand a mechanism of transcranial electrical stimulations and explore the possibility of the application for the treatment of brain diseases and symptom relief [8,9,10,11,12,13]. In addition, based on these results, clinical studies to use tDCS and tACS for clinical brain disease treatment, symptom alleviation, and brain function improvement have been actively progressed [14,15,16,17,18,19]. In particular, the results of several studies indicated that α-band (10 Hz) tACS can be applied to an improvement of sensory/motor function and an up-regulation of the default mode network [20,21,22,23].
Ensuring the safety of medical devices is important for the diagnosis and treatment of diseases through the application of such devices to the human body [24]. In particular, ensuring the safety of transcranial electrical stimulation devices, which contact the scalp and apply electrical stimulation to the brain tissue, is essential for the prevention of unexpected brain damage caused by electromagnetic fields. In a safety evaluation study on tDCS, D. Liebetanz et al. investigated whether brain tissue damage was caused by tDCS with three different currents (I = 0.1, 0.5, and 1.0 mA) and different electrical-stimulation treatment times (T = 30 to 270 min) [25]. The results indicated that if the charge per unit area exceeds a certain threshold (lesion threshold), brain tissue damage occurs, and the extent of damage that occurs when the charge per unit area is above the threshold is proportional to the unit area charge. K. Zhang et al. investigated the safety of 500-μA cathodal tDCS via a brain-function test involving behavioral experiments and histological staining [26]. However, there is a lack of research on the safety of tACS, and safety studies on tACS are needed for the application of tACS to treat brain diseases and the development of transcranial electrical-stimulation-based medical devices.
In this study, we investigated the safety of α-band (10 Hz) tACS using selected parameters (stimulation intensity of 1.0–2.0 mA, frequency of 10 Hz, and treatment time of 20 min). To examine the safety of tACS, alternating current stimulation was applied to rat brains, and the effects and safety of the alternating current stimulation on the brain tissue were evaluated via three methods: (1) macroscale analysis of the damage to the brain tissues through magnetic resonance imaging (MRI), (2) observation of the tissue composition and variations in neuronal cells via hematoxylin and eosin (H&E) and Cresyl violet (Nissl) staining, and (3) measurement of the temperatures between the electrodes and skin in tACS to determine whether the medical device is safe in contact with the skin. Both magnetic resonance brain images and histopathological images of rat brain samples stained by H&E and Cresyl violet indicated that a single, 20 min tACS with a frequency of 10 Hz did not damage the brain tissue or neuronal components. Additionally, infrared thermal imaging indicated that the temperature did not increase above 41 °C even after 20 min of tACS application. This implies that certain conditions of tested tACS and installed electrodes can provide safe tACS, which does not cause damage to brain tissues or neuronal components in the acute phase, with the temperature of the contact regions not exceeding 41 °C.

2. Materials and Methods

2.1. Animal Preparation

This research was permitted by the Institutional Animal Care and Use Committee (IACUC) of Daegu-Gyeongbuk Medical Innovation Foundation with the approval number DGMIF-21062902-00. Six male 7-week-old Sprague Dawley (SD) rats, which were used for safety exploration of tACS, were obtained from Koatech, Pyeongtaek, Korea. The rats were anesthetized via intraperitoneal (IP) injection of 30 mg/kg Zoletil (Zoletil 50 inj., Virbac Korea, Seoul, Korea) and 10 mg/mL Rompun (Rompun inj., Bayer Korea, Seoul, Korea) before the delivery of alternating currents. After anesthesia, we trimmed the hair on the skin of the brain using hair-removal cream (NairTM, Church & Dwight, Ewing, NJ, USA) with povidone-iodine swabs (A17200231, Dongindang, Seoul, Korea). All the rats were allowed free access to food and water.

2.2. Procedures of tACS

After the anesthetization and hair trimming, an anode and a cathode were installed on the head and back, respectively. The anode installed on the head was a 10 mm diameter, gold-coated, disk-shaped electrode (LXEL-SAF-DK-21, Laxtha, Daejeon, Korea), which was installed to the left of the bregma of the rat brain. To increase the conductivity of the disk-type electrodes and minimize the noise, sufficient conductive paste (Elefix; Nihon Kohden, Tokyo, Japan) was applied to the heads of the rats. The cathode installed on the back of the rat was a circular electrode (Kendall Medi-Trace®, Medtronic, Minneapolis, MN, USA) that could be attached to the skin. Two electrodes were connected to a programmable electrical stimulator (Cybermedic, Inc., Iksan, Korea), and the type of electrical stimulation, electrical currents, frequency, and treatment time were entered by a control program. tACS was delivered to the brain of rats under anesthesia. Alternating currents of 1.0, 1.5, and 2.0 mA (at 10 Hz) were transmitted to the rat’s brain (2.0 mA: 2 rats, 1.5 mA: 2 rats, 1.0 mA, one rat, and control (no alternating current was applied): one rat). The radius of the electrode mounted on the head of rats was 5 mm, and thus, the current densities of each alternating current were 2.55 mA/cm2 (2 mA), 1.91 mA/cm2 (1.5 mA), and 1.27 mA/cm2 (1.0 mA). The current density applied in this study is much higher than that of tDCS used in clinical treatments (0.05 mA/cm2) [27], and it is two to four times the current density for high-definition transcranial direct current stimulation [16]. A schematic of the electrode installation and the delivery of tACS is shown in Figure 1a.

2.3. MRI

We applied a 3-T MRI system (MAGNETOM Skyra, Siemens Healthineers, Erlangen, Germany), which was one of the medical imaging instruments in the Fusion Medical Imaging Suite (FMIS) of the K-MEDI hub, to acquire noninvasive and transient macroscale brain images after tACS. Specifically, rats were inserted into a 15-channel knee coil (Siemens Healthineers), and T1- and T2-weighted images of the rat brains were obtained immediately and 48 h after tACS. The head of the rat was placed at a specific position in line with the control point of the knee coil to acquire brain image slices in the same horizontal and vertical axes. The detailed specifications for T1-weighted imaging are as follows: TE (time of echo) = 7.36 ms, TR (time of repetition) = 343.00 ms, and imaging resolution = 0.5 × 0.5 mm. T2-weighted imaging has parameters as follows: TE = 99.00 ms, TR = 7170.00 ms, and imaging resolution is the same as the resolution of T1-weighted images. Post-processing and analysis of DICOM-formed images were performed using a Java-based program (ImageJTM) to compare the T1- and T2-weighted intensities of regions where tACS was applied and on the other side. Specifically, the mean and standard deviation of the intensities were calculated for the 11 × 11-pixel region subjected to transcranial stimulation and the contralateral region in each magnetic resonance brain image.

2.4. Histological Brain Tissue Staining and Analysis

MR images of a rat brain after 48 h of the electrical stimulation were acquired, and each rat was sacrificed. For histopathological assays, H&E and Cresyl violet staining were used to assess the damage to neuronal cells and the cellular morphologies. Each brain slice was fixed in paraformaldehyde and 4% phosphate-buffered saline (PBS, M1176, Biostem, Suwon, Korea) for 24 h. The brain slices were fixed with paraformaldehyde and embedded in paraffin, and the paraffin block was cut to a thickness of 4 µm using a manual microtome (RM23255, Leica Microsystems, Wetzler, Germany). The paraffin-embedded rat brain slices were deparaffinized and hydrated with xylene (Duksan, Ansan, Korea). To investigate the occurrence of brain tissue damage, rat brain slices were stained with an H&E kit (H3502, Vector Laboratories, Inc., Burlingame, CA, USA). Some of the slices were stained with 0.1% Cresyl violet (C5042-10G, Sigma-Aldrich, St. Louis, MO, USA) to analyze the densities of neuronal components. Bright-field images of the stained slices were acquired using an optical slide scanner (Zeiss Axio Scan.Z1; Carl Zeiss, Jena, Germany). For a quantitative analysis of the number of Nissl bodies, five sub-images of 2500 × 2500 pixels were extracted from regions exposed to transcranial stimulations and contralateral regions in a whole-brain slide scan image. A slide scan image post-processing and analysis program (ZEN 3.1, Carl Zeiss) was used for sub-image extraction. For each sub-image, the number of Nissl bodies was determined using the “Threshold” and “Analyze Particles” functions in ImageJTM. A diagram describing the entire process from experimental preparation to histopathological analysis is presented in Figure 1b.

2.5. Temperature Measurements between Electrodes and Skin

An infrared thermal imaging device was employed to measure the temperatures generated on the electrodes before and after alternating current stimulation to ensure that the temperature in the contact regions was maintained below 41 °C, which satisfies the medical device safety standard published by the Ministry of Food and Drug Safety (MFDS) in the Republic of Korea [28] and the IEC (International Electrotechnical Commission) standards for contactable medical device safety [29]. An infrared thermal camera (FLIR C2, FLIR® Systems, Inc., Wilsonville, OR, USA) was used to measure the entire bodies of the rats, including the parts to which the anode and cathode were attached, before and after the electrical stimulation. The measurable temperature range was –10 to 150 °C, and the resolution was 0.1 °C. Quantitative information of measured temperatures was exported using analytical software (FLIR ResearchIR, FLIR® Systems, Inc.).

3. Results and Discussion

3.1. Magnetic Resonance Image-Based Brain Damage Analysis

Referring to a study [25] involving safety verification through the confirmation of brain damage 48 h after tDCS under various conditions, we obtained magnetic resonance images of rat brains immediately and 48 h after 10 Hz tACS was applied. The MRI results are shown in Figure 2. In this study, T1- and T2-weighted magnetic resonance images were obtained. T1-weighted imaging, which has short TE and TR, provides magnetic resonance images with relatively high intensities in the fat of biological tissues. This implies that normal brain tissue appears brighter than the region where brain damage occurs in T1-weighted magnetic resonance images. In contrast, T2-weighted imaging, which has long TE and TR, provides a magnetic resonance image with a higher intensity in water content. Thus, an area where an edema occurs owing to brain tissue damage has a higher intensity than normal brain tissue.
Compared with the results of our previous study [30] on MRI and confirmation of brain damage in a photothrombotic stroke rat model, there was no significant difference in intensities, which would indicate macroscale brain tissue damages, in the T1- or T2-weighted magnetic resonance images for any conditions of tACS applications. In addition, the mean values of the intensities in extracted sub-images with 11 × 11 pixels in tACS-applied and contralateral regions of T1- and T2-weighted magnetic resonance images were calculated to determine whether there was a difference in intensity due to brain damage. The ratios of mean intensity values in the tACS-applied and contralateral regions are compared in Figure 3. Although there was a slight difference in intensity between areas subjected to tACS and the opposite areas for both the T1- and T2-weighted magnetic resonance images, the difference was within one standard deviation. In addition, compared with the control (no electrical current applied), there was no difference in intensity, which would indicate damage to brain tissues, under any conditions of tACS.

3.2. Histological Analysis of Brain Tissues and Neuronal Components

Through H&E staining, the occurrence of damage to the brain tissue was cross-confirmed using magnetic resonance images. In addition, the loss of neuronal components (Nissl bodies) was confirmed by Cresyl violet (Nissl) staining. Slide scan images of H&E- and Cresyl violet-stained brain tissues obtained under each current condition (I = 0.0 (control), 1.0, 1.5, and 2.0 mA) of tACS are shown in Figure 4. Comparing the results of a safety study on tDCS [25] and our previous study on rabbit brain tissues in which brain damage was induced by a photothrombotic stroke [31], there was no area where the brain tissue density decreased under all conditions of tACS applications. In addition, slide scan images of Cresyl violet-stained brain tissue indicated no loss of Nissl bodies caused by brain damage from tACS.
For a quantitative analysis of the neuronal components, we extracted five sub-images with 2500 × 2500 pixels (=660 × 660 μm2) each from the area where tACS was applied and the contralateral area in slide scan images, and the number of Nissl bodies in the sub-images was calculated, as shown in Figure 5. Although the numbers of Nissl bodies differed between the region to which tACS was applied and the contralateral region, the difference was not significant. Specifically, there was a difference in the number of Nissl bodies between the two regions in all the experimental cases, including the control (no current), but the deviation was approximately 10%; thus, there did not appear to be a significant reduction in neuronal components due to brain damage.

3.3. Temperature Measurements of Electrodes before and after tACS

Figure 6 shows thermal images of rats measured before and immediately after 20 min of application of 10 Hz tACS with an electrical current of 1.5 and 2.0 mA. It was confirmed that the regions where an anode or a cathode was located on the head and back of rats had higher temperatures than other regions. However, even after 20 min of tACS application, the temperature did not increase above 41 °C. This implies that tACS using the electrical stimulator and electrodes employed in this study does not violate the regulation for the temperature conditions of skin-contacting medical devices.

3.4. Discussion

In this study, the safety of tACS with specific parameters in brain tissue and neuronal components was confirmed by MRI and histopathological imaging of H&E- and Cresyl violet-stained brain slide samples. Acquired magnetic resonance brain images and histopathological images indicated that a single, 20 min tACS with a frequency of 10 Hz did not cause damage to brain tissue or neuronal components in the acute phase. In addition, it was confirmed that tACS under these conditions did not increase the surface temperature above 41 °C in the contact regions of both the anode and the cathode. In this preliminary study, the safety was evaluated by applying specific alternating current stimulation conditions, and the techniques developed in this research can be employed to ensure the safety of tACS at other frequencies, e.g., the γ-band (40 Hz), and tRNS [32,33,34,35,36,37]. The specifications of the electrode (contact area, material, immersion media, etc.), as well as the frequency, are important factors in transcranial electrical stimulations; therefore, the safety of other electrodes should be assessed.
This research corresponds to a preliminary study that identified an occurrence of acute brain tissue damage and safety of tACS for several current intensities, a specific frequency (10 Hz), and a specific stimulation delivery time (20 min), and thus it has the following limitations. To ensure sufficient safety and usability of tACS for clinical applications, parametric and comparative studies with varying current densities, frequencies, treatment times, and types of electrodes are required. In addition, this study corresponds to a preliminary study to investigate the occurrence of brain tissue damage in a short period of time (48 h). For confirmation of the long-term safety of tACS, longitudinal observational studies, including behavior experiments, are required for tACS with each condition. In this research, the minimum number of rats for each condition of tACS was used to confirm the occurrence of brain tissue damage. To secure safety under experimental conditions that characteristics between individual rats are excluded as much as possible, it is determined that observational case-control studies with a large number of animals are required. For the application of tACS to treat a specific brain disease, in vivo studies are needed to simultaneously secure both the safety and therapeutic effectiveness of tACS for animal models with a specific brain disease.
In this study, MRI, which is a noninvasive and longitudinal brain imaging method, and end-point histological imaging of brain tissues (H&E) and neuronal components (Nissl) were employed for safety assessments of tACS. The results were derived from the most basic noninvasive imaging and histopathological staining used to determine tissue damages, and studies to confirm safety for individual neuronal components in smaller units than tissues are needed. To obtain information on the safety of specific neuronal cells (neurons, astrocytes, glial cells) and biological molecules in the brain, additional immunostaining assays are required. Molecular probes capable of labeling neurons (NeuN), astrocytes (glial fibrillary acidic protein (GFAP)), glial cells (Iba-1), activated macrophages (ED-1), and other biomolecules related to neural activity can effectively acquire information on the safety of tACS in single cells and biomolecules [13,26,38,39,40,41]. These are biomarkers employed in previous studies to ensure the safety and effectiveness of tDCS. As a representative study, L. Cherchi et al. confirmed that two 20 min tDCS treatments with an interval of 20 min had therapeutic effectiveness and safety in stroke-induced mice by analyzing changes in both astrocyte and microglia [42].
Temperature measurement techniques for regions to which electrodes are attached can be employed to proactively analyze temperature variations caused by other contact types and energy-transfer medical devices. This can contribute to the development of skin-contacting medical devices that satisfy regulations.

4. Concluding Remarks

The results of magnetic resonance brain imaging, histological assays, and temperature measurements of electrode contact regions confirmed that the conditions applied in this study provide safe tACS without damaging brain tissues and neuronal components in the acute phase. When the techniques used in this research, parametric studies, and additional assessments of biomolecules are combined, it is expected that the optimal transcranial electrical stimulation conditions for safety at the preclinical stage can be identified.

Author Contributions

Conceptualization, S.S.O. and J.-r.C.; methodology, S.S.O., S.-H.A., and J.-r.C.; in vivo experiment, S.S.O., Y.B.L., and J.-r.C.; histopathological sample preparation, Y.B.L. and J.S.J.; analysis and validation, S.S.O., J.S.J., and J.-r.C.; writing—original draft preparation, S.S.O. and J.-r.C.; writing—review and editing, S.S.O., S.-H.A., and J.-r.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI17C1501) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science & ICT (NRF-2020R1C1C1012230).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Daegu-Gyeongbuk Medical Innovation Foundation with the approval number DGMIF-21062902-00 (Date of approval: 21 June 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available with a presentation of this article as a reference article with a statement of article information (a title, a journal name, and a publisher).

Conflicts of Interest

The authors report no conflicts of interest. Furthermore, Cybermedic, Inc. has no influence on the design of the study, data collection, analysis and interpretation of the data, or on the writing of the manuscript.

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Figure 1. (a) Schematic of the installation of electrodes and the delivery of tACS. (b) Procedures for the safety assessment of tACS.
Figure 1. (a) Schematic of the installation of electrodes and the delivery of tACS. (b) Procedures for the safety assessment of tACS.
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Figure 2. Longitudinal T1- and T2-weighted magnetic resonance images of rat brains acquired immediately (0 h) and 48 h after tACS with a frequency of 10 Hz and a treatment time of 20 min. Yellow boxes in MR images indicate regions segmented for quantitative analysis of mean intensity values. (T: Transcranial alternating current stimulation-applied region, C: Contralateral region).
Figure 2. Longitudinal T1- and T2-weighted magnetic resonance images of rat brains acquired immediately (0 h) and 48 h after tACS with a frequency of 10 Hz and a treatment time of 20 min. Yellow boxes in MR images indicate regions segmented for quantitative analysis of mean intensity values. (T: Transcranial alternating current stimulation-applied region, C: Contralateral region).
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Figure 3. Ratios of the mean intensity values in tACS-applied and contralateral regions from (a) T1- and (b) T2-weighted magnetic resonance images. Although there was a slight difference in intensity between areas subjected to tACS and the opposite areas in both the T1- and T2-weighted magnetic resonance images, the difference was within a level of standard deviation.
Figure 3. Ratios of the mean intensity values in tACS-applied and contralateral regions from (a) T1- and (b) T2-weighted magnetic resonance images. Although there was a slight difference in intensity between areas subjected to tACS and the opposite areas in both the T1- and T2-weighted magnetic resonance images, the difference was within a level of standard deviation.
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Figure 4. Histopathological slide scan images of H&E and Cresyl violet-stained brain tissue slides under each current condition (I = 0.0 (control), 1.0, 1.5, and 2.0 mA) of 10 Hz tACS. Red boxes indicate regions of sub-images segmented for quantitative analysis of a number of Nissl bodies. (T: Transcranial alternating current stimulation-applied region, C: Contralateral region).
Figure 4. Histopathological slide scan images of H&E and Cresyl violet-stained brain tissue slides under each current condition (I = 0.0 (control), 1.0, 1.5, and 2.0 mA) of 10 Hz tACS. Red boxes indicate regions of sub-images segmented for quantitative analysis of a number of Nissl bodies. (T: Transcranial alternating current stimulation-applied region, C: Contralateral region).
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Figure 5. Quantitative comparison of the numbers of Nissl bodies (neuronal components) in sub-images of Cresyl violet-stained brain slide scan images. Although there was a difference in the number of Nissl bodies between the two regions in all the experimental cases, including the control (no current), the deviation was approximately 10%, indicating that there was no significant reduction in neuronal components due to brain damage.
Figure 5. Quantitative comparison of the numbers of Nissl bodies (neuronal components) in sub-images of Cresyl violet-stained brain slide scan images. Although there was a difference in the number of Nissl bodies between the two regions in all the experimental cases, including the control (no current), the deviation was approximately 10%, indicating that there was no significant reduction in neuronal components due to brain damage.
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Figure 6. Thermal images of rats measured before and immediately after 20 min applications of 10 Hz tACS with an electrical current of 1.5 or 2.0 mA. Regions where an anode or a cathode was located on the heads and backs of rats had higher temperatures than other regions, but the temperature did not increase above 41 °C even after 20 min of tACS. The average, the maximum, and the minimum temperatures of regions where the anode and the cathode were attached are described on the right side of the thermal images.
Figure 6. Thermal images of rats measured before and immediately after 20 min applications of 10 Hz tACS with an electrical current of 1.5 or 2.0 mA. Regions where an anode or a cathode was located on the heads and backs of rats had higher temperatures than other regions, but the temperature did not increase above 41 °C even after 20 min of tACS. The average, the maximum, and the minimum temperatures of regions where the anode and the cathode were attached are described on the right side of the thermal images.
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Oh, S.S.; Lee, Y.B.; Jeon, J.S.; An, S.-H.; Choi, J.-r. Preliminary Study on Safety Assessment of 10 Hz Transcranial Alternating Current Stimulation in Rat Brain. Appl. Sci. 2022, 12, 5299. https://doi.org/10.3390/app12115299

AMA Style

Oh SS, Lee YB, Jeon JS, An S-H, Choi J-r. Preliminary Study on Safety Assessment of 10 Hz Transcranial Alternating Current Stimulation in Rat Brain. Applied Sciences. 2022; 12(11):5299. https://doi.org/10.3390/app12115299

Chicago/Turabian Style

Oh, Sung Suk, Yoon Bum Lee, Jae Sun Jeon, Sang-Hyun An, and Jong-ryul Choi. 2022. "Preliminary Study on Safety Assessment of 10 Hz Transcranial Alternating Current Stimulation in Rat Brain" Applied Sciences 12, no. 11: 5299. https://doi.org/10.3390/app12115299

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