## 3 Results and Discussion
### 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 \({}^{\circ}\)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 \(\gamma\)-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, [PERSON] 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.