Energy-Dependent Cortical Injury Thresholds in High-Frequency Transcortical Electrical Stimulation: A Biophysical Study in a Rat Model

Energy-Dependent Cortical Injury Thresholds in High-Frequency Transcortical Electrical Stimulation: A Biophysical Study in a Rat Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Energy-Dependent Cortical Injury Thresholds in High-Frequency Transcortical Electrical Stimulation: A Biophysical Study in a Rat Model Ridzky Firmansyah Hardian, Kunihiko Kodama, Kohei Kanaya, Tetsuya Goto, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9558913/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The biophysical determinants of cortical tissue injury during brief, high-frequency transcortical electrical stimulation remain incompletely understood. Traditional safety criteria derived from chronic, low-frequency paradigms emphasize total charge, but whether charge or stimulation energy is the primary predictor of injury under high-frequency conditions—such as those used for intraoperative motor evoked potential (MEP) monitoring—is unclear. Thirty-two Sprague-Dawley rats (8 groups of 4) received monophasic anodal transcortical pulse trains with varying current, pulse duration, repetition number, and interstimulation interval. Maximum cortical lesion depth was measured histologically, and the contributions of total charge (Q = I × t) and relative stimulation energy (W ∝ I²t) were dissociated through controlled group comparisons and multiple linear regression. Higher stimulation current (p = 0.04) and greater repetition number (p = 0.005) significantly increased lesion depth. Multiple regression identified total stimulation energy as the only significant independent predictor (p = 0.008), whereas total charge was not (p = 0.677). With equivalent total charge, higher-energy stimulation tended to produce deeper lesions (209 ± 67 vs. 125 ± 101 µm, p = 0.25); when total energy was equalized despite a 2.5-fold difference in total charge, lesion depths were nearly identical (p = 1.0). Under high-frequency conditions, cortical injury depth is governed primarily by total energy deposition rather than total charge, consistent with biophysical mechanisms involving resistive heating and electrolytic processes at the electrode–tissue interface. These findings provide a mechanistic framework for understanding electrical tissue injury under MEP-relevant stimulation regimes. transcortical stimulation cortical lesion depth biophysics resistive heating rat model Figures Figure 1 Figure 2 Figure 3 Introduction Intraoperative motor evoked potential (MEP) monitoring with transcortical stimulation is widely used to preserve motor pathways during brain surgery (Taniguchi et al. 1993 ; Krieg et al. 2012 ; Ichikawa et al. 2010 ). The technique relies on brief trains of high-frequency pulses (typically several hundred Hz) delivered at intensities often exceeding those used in chronic cortical stimulation paradigms (Pechstein et al. 1996 ; Oinuma et al. 2007 ). Despite the widespread clinical use of this stimulation regime, the biophysical determinants of cortical tissue injury under such conditions remain poorly defined. Traditional safety criteria for cortical stimulation, including Shannon’s model, emphasize charge density per phase and total charge density as primary predictors of neural injury (Shannon 1992 ; Cogan et al. 2016 ; Yuen et al. 1981 ; McCreery et al. 1990 ; Gordon et al. 1990 ; MacDonald 2002 ). However, these criteria were derived from chronic, low-frequency paradigms in which stimulation was delivered continuously over hours to days (Yuen et al. 1981 ; McCreery et al. 1990 ; Harnack et al. 2004 ). The brief, high-frequency, repetitive trains used for intraoperative MEP differ fundamentally from these conditions in their temporal pattern of energy delivery and in the instantaneous power dissipated in tissue. Whether charge-based safety criteria adequately capture the biophysical relationship between stimulation parameters and the resulting volumetric tissue damage in this regime remains an open question. In the present study, we provide a mechanistic framework by evaluating the relative contributions of electrical charge (Q = I × t) and stimulation energy (W ∝ I²t) to the depth of cortical lesions in a rat model under MEP-relevant high-frequency stimulation. By experimentally dissociating these two parameters—comparing groups with matched total charge but different total energy, and vice versa—we sought to identify the primary biophysical driver of tissue injury and to clarify the underlying physical mechanisms, including resistive heating and anodic electrolysis. Methods Thirty-two 8- to 12-week-old male Sprague-Dawley rats (weight 350–450 grams) were used for the experiments. The rats were provided by the vivarium of Shinshu University School of Medicine. The animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by Shinshu University's Ethical Review Board. The rat experiment was approved by the Ethical Committee of Shinshu University (approval no. 200024). All procedures were designed in accordance with the 3R principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and to reduce suffering. After induction of general anesthesia with intraperitoneal pentobarbital (60 mg/kg), all efforts were made to minimize pain and distress. The head was fixed to a rat head-fixation device. Bilateral frontal craniectomies with a diameter of 10 mm were performed, and the dura mater was exposed. The bilateral dura mater was incised circumferentially to expose the cerebral cortex. The arachnoid membrane and the cerebral cortex were kept intact. A pair of platinum electrodes with a diameter of 2 mm (Unique Medical Co. Ltd, Japan) were placed on the cerebral cortex bilaterally (Fig. 1 ). Anodal monopolar high-frequency monophasic stimulation, which is a standard MEP stimulation method (Szelényi et al. 2007 ), was used to deliver electrical stimulation to the cerebral cortex. The placement of the stimulating electrode ensured that stimulation was delivered through the uninjured cortex. Neuropack Σ® (Nihon Kohden, Japan) with a constant-current stimulator was used as a stimulation machine. A five-pulse train stimulation with a pulse frequency of 500 Hz, an interstimulation interval (ISI) of 2 ms, a current of 20 mA or 50 mA, a duration of 0.2 ms or 0.05 ms, an interval between each stimulation session of 1s or 10s, and stimulus repetition of 0 (control), 10x, 100x, or 625x was delivered. This stimulation protocol resulted in 8 groups of rats (4 rats per group) with various combinations of stimulation parameters (Table 1 ). Table 1 Stimulation parameter for each group of the experiments Rats group Current (mA) Duration (ms) Interstimulation interval (s) Stimulation number (n) Total charge (µC) Relative energy (I 2 t, arbitrary units) Mean lesion depth (µm) 1 50 0.2 1 0 0 0 0 2 50 0.2 1 10 500 25 0 3 50 0.2 1 100 5000 250 209 ± 67 4 20 0.2 1 100 2000 40 97 ± 33 5 50 0.05 1 100 1250 62.5 157 ± 141 6 50 0.2 10 100 5000 250 183 ± 36 7 20 0.2 1 625 12500 250 211 ± 59 8 20 0.5 1 100 5000 100 125 ± 101 Eight experimental groups were designed to evaluate the following comparisons: (1) the effect of stimulation number (groups 1–3 at 50 mA, and groups 4 and 7 at 20 mA); (2) the effect of stimulation current (groups 3 vs. 4, 50 mA vs. 20 mA, 100 repetitions); (3) the effect of stimulation duration (groups 3 vs. 5 at 50 mA, and groups 4 vs. 8 at 20 mA); (4) the effect of interstimulation interval (groups 3 vs. 6, 1 s vs. 10 s); (5) same total charge with different total energy (groups 3 vs. 8); (6) same total energy with different total charge (groups 3 vs. 7); and (7) the overall association between total charge or energy and lesion depth across all groups. Full stimulation parameters for each group are provided in Table 1 . Total stimulation charge (Q) was defined as Q = I × t, where I is the stimulation current, and t is the total stimulation time (pulse duration × number of stimulations). Stimulation energy was expressed as proportional to I²t (W ∝ I²t), assuming relatively constant tissue resistance across experimental conditions; accordingly, relative differences in I²t were used as a surrogate for energy comparison across groups. The platinum electrodes were removed immediately after stimulation. Animals were humanely euthanized by an overdose of pentobarbital (150 mg/kg, intraperitoneally) in accordance with institutional and international guidelines. Whole-body perfusion fixation with 9% formaldehyde was performed, and then the removed brain was formalin-fixed. After appropriate fixation, the brain was vertically and coronally sectioned at the center position of the anodal electrode and then paraffin-fixed. A brain preparation was created with a thickness of 3.0 µm from the stimulated brain surface and stained with hematoxylin and eosin. Under an optical microscope, the maximum depth of the lesion in the cerebral cortex of the anodal stimulation was measured. The depth of the cerebral cortex lesion between the groups of rats was compared using the Mann-Whitney and Kruskal-Wallis tests. The correlation between stimulation charge and energy with the lesion depth was statistically analyzed with multiple linear regression. The p-value was considered significant if p < 0.05. Statistical analysis was performed using SPSS version 21. Results Transcortical stimulation was delivered to all groups except the control (group 1) according to the research protocol. The electrode impedance was approximately 1 kΩ. The brain tissues under the anodal electrode were observed under an optical microscope. There were no histological changes found in rats in groups 1 (control) and 2 (Fig. 2 A). A downwardly directed convex hemispherical lesion was observed from the surface of the cortex to a deeper part of the cortex, consistent with the part of the electrode in contact with the disc plane in the rats in groups 3–8 (Fig. 2 B). Tissue vacuolation, swelling, and microbleeding were confirmed inside the lesions (Fig. 2 C). The lesion depth, which was the distance between the cortical surface and the deepest part of the lesion, was measured. The lesion depth ranged from 0 µm to 296 µm, with an average of 122.8 ± 101.3 µm. The average lesion depth for each group with their respective parameters of electrical stimulation is shown in Table 1 . The main findings are summarized below. Regarding stimulation number, lesion depth increased significantly with greater repetition: groups 1 and 2 showed no lesion (0 µm), whereas group 3 reached 209 ± 67 µm (p = 0.005 for groups 1–3), and group 7 reached 211 ± 59 µm compared with 97 ± 33 µm in group 4 (p = 0.02). Higher stimulation current also produced significantly greater lesion depth: 209 ± 67 µm (group 3, 50 mA) vs. 97 ± 33 µm (group 4, 20 mA; p = 0.04). All comparisons are summarized in Table 2 . Table 2 Comparison of different stimulation parameters toward lesion depth Compared parameters Shared parameters Total charge (µC) Rat group Mean lesion depth (µm) p Value Stimulation number 1 (times) SC:50 mA, SD: 0.2 ms, SI: 1s 0.005 0x 0 Group 1 0 10x 500 Group 2 0 100x 5000 Group 3 209 ± 67 Stimulation number 2 (times) SC:20 mA, SD: 0.2 ms, SI: 1s 0.02 100x 2000 Group 4 97 ± 33 625x 12500 Group 7 211 ± 59 Stimulation current (mA) SD: 0.2 ms, SN:100x, SI: 1s 0.04 20 mA 2000 Group 4 97 ± 33 50 mA 5000 Group 3 209 ± 67 Stimulation duration 1 (ms) SC: 50 mA, SN: 100x, SI: 1s 0.56 0.05 ms 1250 Group 5 157 ± 141 0.2 ms 5000 Group 3 209 ± 67 Stimulation duration 2 (ms) SC: 20 mA, SN: 100x, SI: 1s 0.25 0.2 ms 2000 Group 4 97 ± 33 0.5 ms 5000 Group 8 125 ± 101 Stimulation interval (s) SC: 50 mA, SD: 0.2 ms, SN: 100x 0.09 1s 5000 Group 3 209 ± 67 10s 5000 Group 6 183 ± 36 SC: stimulation current, SD: stimulation duration, SI: stimulation interval, SN: stimulation number In contrast, neither pulse duration nor interstimulation interval significantly affected lesion depth. Increasing duration from 0.05 to 0.2 ms (groups 5 vs. 3: 157 ± 141 vs. 209 ± 67 µm, p = 0.56) and from 0.2 to 0.5 ms (groups 4 vs. 8: 97 ± 33 vs. 125 ± 101 µm, p = 0.25) produced numerically larger lesions, but the differences did not reach significance. Similarly, extending the interstimulation interval from 1 to 10 s (groups 3 vs. 6: 209 ± 67 vs. 183 ± 36 µm, p = 0.09) showed a trend toward smaller lesions without reaching significance (Table 2 ). When total charge was held constant, but total energy differed (groups 3 vs. 8: 5,000 µC for both; relative energy I²t: 250 vs. 100 [arbitrary units]), mean lesion depth was numerically greater in the higher-energy group (209 ± 67 vs. 125 ± 101 µm, p = 0.25; Fig. 3 , left). Conversely, when total energy was equalized but total charge differed 2.5-fold (groups 3 vs. 7: 5,000 vs. 12,500 µC; relative energy I²t: both 250 [arbitrary units]), mean lesion depths were virtually identical (209 ± 67 vs. 211 ± 59 µm, p = 1.0; Fig. 3 , right). Multiple linear regression analysis across all groups yielded the equation: lesion depth = 39.24 + 0.02 × Q + 0.61 × W. Total stimulation energy was the only significant independent predictor (p = 0.008), whereas total stimulation charge was not (p = 0.677). Discussion The present study examined the relative biophysical contributions of electrical charge and stimulation energy to cortical lesion depth under MEP-relevant high-frequency stimulation. Stimulation current and the number of stimulations were the parameters most consistently associated with greater lesion depth. Most importantly, controlled comparisons between groups with equivalent total charge but differing total energy demonstrated that parameters contributing to total stimulation energy (W ∝ I²t) play a greater role in determining lesion depth than total charge (Q = I × t) alone, a pattern further supported by multiple regression in which total energy was the only significant independent predictor (p = 0.008) while total charge was not (p = 0.677). Although the small group sizes (n = 4 per group) limited statistical power for detecting subtle effects of pulse duration or interstimulation interval, the consistency of the energy-dependent pattern across multiple controlled comparisons supports the validity of this mechanistic framework and identifies energy deposition as a dominant biophysical driver of cortical injury under these conditions. Electrical tissue damage beneath a stimulating electrode is histologically characterized by neuronal vacuolation, edema, disruption of cytoarchitecture, vasodilation, and microhemorrhage (Yuen et al. 1981 ; McCreery et al. 1990 ). Multiple physicochemical mechanisms have been implicated, including gas evolution, local pH changes, resistive heating, and direct electrochemical effects on cellular membranes (MacDonald 2002 ; Berendson and Simonsson 1994 ; Butterwick et al. 2007 ). These factors accumulate with increasing stimulation intensity (Yuen et al. 1981 ; Berendson and Simonsson 1994 ). At the anode in particular, oxidation reactions produce local acidification, which is considered a primary driver of tissue injury (Berendson and Simonsson 1994 ). The ohmic voltage drop across the electrode–tissue interface contributes relatively little to tissue damage, as most of the applied charge is consumed in charging the electrical double layer rather than driving faradaic reactions. Collectively, these mechanisms converge through tissue electrolysis to produce the observed lesion pattern. Prior work on chronic cortical stimulation has emphasized charge density per phase and total charge density as key determinants of neural injury, with McCreery et al. demonstrating a synergistic relationship between charge density and charge per pulse (Yuen et al. 1981 ; McCreery et al. 1990 ). Shannon's model similarly highlighted charge density as a primary predictor of damage thresholds (Cogan et al. 2016 ; Yuen et al. 1981 ; McCreery et al. 1990 ; Gordon et al. 1990 ; MacDonald 2002 ; Shannon 1992 ). However, those safety criteria were derived from chronic, low-frequency paradigms and may not directly translate to the brief, high-frequency, repetitive stimulation used for intraoperative MEP monitoring (Pechstein et al. 1996 ; Oinuma et al. 2007 ). Furthermore, inconsistencies in the relationship between charge density and neural damage across prior studies suggest that additional factors beyond charge alone may contribute to electrical injury during MEP-relevant stimulation. The dominance of energy over charge in tissue injury The present results suggest that, under MEP-relevant stimulation conditions, total energy may be a more informative predictor of lesion depth than total charge. In the key controlled comparison (Groups 3 vs. 8), both groups received identical total charge (5,000 µC), yet the group with higher total energy (relative energy I²t: 250 vs. 100 [arbitrary units]) showed a numerically greater mean lesion depth (209 ± 67 µm vs. 125 ± 101 µm, p = 0.25). Conversely, when total energy was held constant while total charge differed by 2.5-fold (Groups 3 vs. 7: 5,000 µC vs. 12,500 µC), mean lesion depths were virtually identical (209 ± 67 µm vs. 211 ± 59 µm, p = 1.0). Although neither comparison reached statistical significance—likely reflecting the limited sample size—the consistent directional pattern across both experiments supports the hypothesis that energy-related parameters drive lesion depth more than charge-related parameters under these conditions. Biophysical mechanisms: resistive heating and electrolysis A plausible mechanistic basis for this energy dependence lies in resistive heating. Because power dissipation in tissue scales with the square of current (P = I²R), a higher stimulation current generates disproportionately more heat per unit time than a lower current delivering equivalent charge over a longer pulse duration. This I² dependence of energy deposition may explain why stimulation current emerged as a significant predictor of lesion depth, while pulse duration—despite influencing total charge—did not. The pulse durations tested in the present study (0.05–0.5 ms) span a range that overlaps with the chronaxie of cortical gray matter (0.2–0.7 ms). Charge delivery near chronaxie is considered physiologically efficient for neural activation (Ranck 1975 ; Abalkhail et al. 2017 ), but this same range may limit the independent contribution of pulse duration to electrochemical injury when current is held constant. The present results are consistent with this interpretation: in comparisons where total charge was equalized between groups but current differed (Groups 4 vs. 3 and Groups 4 vs. 8), lesion depth was consistently greater in the higher-current group, further suggesting that current magnitude—and by extension, energy—may be more relevant than pulse duration to the depth of cortical injury. The present findings may also help reconcile apparent discrepancies between our results and prior charge-density-based models (Cogan et al. 2016 ; Yuen et al. 1981 ; McCreery et al. 1990 ). Charge density tends to be highest at the electrode periphery, which may contribute predominantly to surface morphological damage rather than to the depth of injury measured here. Stimulation energy, by contrast, may better reflect the volumetric extent of tissue heating and electrolytic injury beneath the electrode. This distinction—charge density as a predictor of surface morphological severity versus energy as a predictor of lesion depth or volume—may help reconcile apparent discrepancies between prior histological studies and the present depth-based measurements. Implications for safer stimulation protocols These findings carry several practical implications for the safe conduct of intraoperative MEP monitoring, bearing in mind their exploratory nature. When it is necessary to increase stimulation intensity—for example, in patients with preoperative motor deficits, high stimulation thresholds, or monitoring of muscles with inherently high thresholds such as lower extremity musculature (Hardian et al. 2021 )—the present data suggest that increasing pulse duration rather than stimulation current may represent the safer strategy, as it increases total charge with a smaller proportional increase in energy. Keeping the pulse duration near the chronaxie of the target tissue would be expected to maintain stimulation efficiency while minimizing energy deposition (Abalkhail et al. 2017 ). Second, the strong influence of stimulation number on lesion depth underscores the importance of threshold-based stimulation protocols that minimize unnecessary repetitions (Abboud et al. 2016 ; Calancie et al. 2001 ; Hardian et al. 2019 ). The threshold was defined in the present study as the lowest intensity yielding a reproducible MEP response with amplitude ≥ 20 µV and appropriate latency (Hardian et al. 2021 ; Goto et al. 2010 ; Kanaya et al. 2019a ), an approach that limits cumulative energy deposition while preserving monitoring sensitivity. When no MEP response is obtained, the stimulation number must be increased, though a certain additional margin may be acceptable to ensure reliable clinical monitoring. It is important to note that the stimulation parameters and electrode geometry used here are not directly representative of clinical practice. Standard intraoperative transcortical electrodes have a diameter of approximately 5 mm, whereas the 2 mm electrodes used in the present study are smaller. For equivalent stimulation current (20 mA), the charge density per phase beneath a 5 mm electrode is approximately 1/15 that of a 2 mm electrode, and if tissue change scales proportionally with energy, the effective damage would be expected to be substantially reduced (Goto et al. 2018 ; Kanaya et al. 2019b ). Furthermore, in clinical practice, stimulation is typically delivered at intervals of several seconds to minutes rather than the 1-second interval used here, allowing greater thermal and electrochemical dissipation between trials. These differences suggest that tissue injury under typical clinical conditions would be substantially lower than that observed experimentally, consistent with the overall clinical safety record of transcortical MEP monitoring. Accordingly, the present findings should be interpreted as providing a mechanistic framework rather than a direct representation of clinical conditions, and caution should be exercised when extrapolating to the clinical setting (Taniguchi et al. 1993 ; Neuloh and Schramm 2009 ). Nevertheless, the present results serve as a reminder that apparently safe stimulation intensities may produce histological changes at the site of electrode contact, particularly under conditions of prolonged surgery or a high number of stimulation trials, and that minimizing total stimulation energy throughout the procedure remains prudent. Study limitations and exploratory nature Several limitations must be acknowledged. First, rat and human cortex differ in geometric organization and cellular density, and the tolerance of human cortex to electrical stimulation may not be identical to that of the rat (Hodge et al. 2019 ; DeFelipe 2011 ). Nevertheless, the fundamental cytoarchitecture and cellular constituents of the mammalian cerebral cortex are broadly conserved, supporting the relevance of the rat model for mechanistic investigation. Second, the present study measured lesion depth as a surrogate for injury severity. Whether the observed histological changes translate into functional motor deficits remains unknown and warrants dedicated investigation. Third, only a single electrode size was employed, precluding direct assessment of how electrode surface area modulates the relationship between energy, charge density, and injury depth. Variability in electrode contact area—due to intraoperative handling or gas formation at the electrode–tissue interface—represents an additional source of uncertainty in both experimental and clinical settings. Fourth, the small group size (n = 4 per group) substantially limits statistical power and generalizability. The non-significant p-values observed in several comparisons should therefore be interpreted with caution; they may reflect insufficient power to detect true differences rather than the absence of an effect. Replication with larger cohorts and a broader range of stimulation parameters is needed to confirm these observations. Finally, because brain tissue is electrically inhomogeneous and anisotropic (Aström et al. 2012 ), the simplified assumption of constant resistance used to calculate energy may introduce systematic error. In this study, stimulation energy was treated as a relative index proportional to I²t, rather than an absolute quantity, because tissue resistance was not directly measured. Accordingly, the energy values reported here should be interpreted as relative comparisons between groups rather than true physical energy values. More refined models incorporating direct tissue impedance measurements would be required to validate this approach and strengthen future analyses. Conclusions Under high-frequency transcortical stimulation conditions, the depth of cortical injury is more closely associated with total stimulation energy (W ∝ I²t) than with total electrical charge alone, consistent with biophysical mechanisms involving resistive heating and electrolytic processes at the electrode–tissue interface. Stimulation current and the number of stimulations are the dominant determinants of energy deposition and, consequently, of lesion depth, whereas pulse duration and interstimulation interval play comparatively minor roles within the parameter ranges examined. These findings provide a mechanistic framework that may inform safer stimulation protocols: when stronger stimulation output is required, increasing pulse duration at constant current may be preferable to increasing current itself, as it raises total charge with a smaller proportional rise in energy. Replication in larger cohorts and with direct tissue impedance measurements is needed to establish quantitative thresholds across the parameter space. Declarations Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by Shinshu University’s Ethical Review Board, and were approved by the Ethical Committee of Shinshu University (approval no. 200024). All procedures were designed in accordance with the 3R principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and to reduce suffering. The reporting of this study conforms to the ARRIVE guidelines for animal pre-clinical research. Consent to participate Not applicable. This study did not involve human participants. Consent for publication Not applicable. This study did not involve human participants. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution Ridzky Firmansyah Hardian: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Kunihiko Kodama: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Kohei Kanaya: Conceptualization, Methodology, Formal analysis, Writing – review & editing, Supervision. Tetsuya Goto: Methodology, Writing – review & editing, Supervision. Kazuhiro Hongo: Writing – review & editing, Supervision. Tetsuyoshi Horiuchi: Writing – review & editing, Supervision, Project administration. All authors read and approved the final manuscript. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. 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IEEE Trans Biomed Eng 39:424–426. https://doi.org/10.1109/10.126616 Szelényi A, Kothbauer KF, Deletis V (2007) Transcranial electric stimulation for intraoperative motor evoked potential monitoring: stimulation parameters and electrode montages. Clin Neurophysiol 118:1586–1595. https://doi.org/10.1016/j.clinph.2007.04.008 Taniguchi M, Cedzich C, Schramm J (1993) Modification of cortical stimulation for motor evoked potentials under general anesthesia: technical description. Neurosurgery 32:219–226. https://doi.org/10.1227/00006123-199302000-00011 Yuen TG, Agnew WF, Bullara LA, Jacques S, McCreery DB (1981) Histological evaluation of neural damage from electrical stimulation: considerations for the selection of parameters for clinical application. Neurosurgery 9:292–299 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9558913","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638302313,"identity":"ed90d962-a1fe-450e-982a-4b3a84d1208d","order_by":0,"name":"Ridzky Firmansyah Hardian","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ridzky","middleName":"Firmansyah","lastName":"Hardian","suffix":""},{"id":638302315,"identity":"83123bbd-8676-44e1-8203-04ee2bef5b2f","order_by":1,"name":"Kunihiko Kodama","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kunihiko","middleName":"","lastName":"Kodama","suffix":""},{"id":638302321,"identity":"360a5bea-2cd1-4c35-ba66-241e63e88b3d","order_by":2,"name":"Kohei Kanaya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACCQhlw2DAzMDAjC6MT0sa6VoOMxgwIGvBByTbzx78zFNz3t6cnffw58I2hsQG9sMPGCx34NYizZOXLM1z7Hbizma+NOmZIC08aQYMkmdwa5FjyDGQ5mG7nWBwmMeMmbftf2IDQw7Q8jY8WvjfGP/m+XfOHqjF+DMvyBb+N/i1SEvkmEnzth1g3HCYx0AarEWCgC2SM96YWc7tS04EajGT5jnHYNwm8czgAD6/SJzPMb7x5pudvcH5M8afecoYZPv5kx8+lsQTYiDAxIPMYwPiw5IN+LUw/sAQ+UhAyygYBaNgFIwoAAAPH0cYYXfXvgAAAABJRU5ErkJggg==","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kohei","middleName":"","lastName":"Kanaya","suffix":""},{"id":638302327,"identity":"ad6d43d7-fed2-478a-8d32-44a4e82fd1d0","order_by":3,"name":"Tetsuya Goto","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tetsuya","middleName":"","lastName":"Goto","suffix":""},{"id":638302328,"identity":"1f206b02-6afc-41eb-9cb3-4f66c7358a16","order_by":4,"name":"Kazuhiro Hongo","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Hongo","suffix":""},{"id":638302330,"identity":"cc3395fa-542e-434a-bad4-83404719ecf8","order_by":5,"name":"Tetsuyoshi Horiuchi","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tetsuyoshi","middleName":"","lastName":"Horiuchi","suffix":""}],"badges":[],"createdAt":"2026-04-29 00:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9558913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9558913/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109205252,"identity":"e5da9ece-7b90-43ba-9628-595fab1d4944","added_by":"auto","created_at":"2026-05-13 15:03:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27377818,"visible":true,"origin":"","legend":"\u003cp\u003ePlatinum electrodes (arrows) with adiameter of 2 mm wereplaced on the bilateral cerebral cortex of the rat \u003cstrong\u003e(left)\u003c/strong\u003e. The electrode attachment to the cerebral cortex was shown in a magnified photo (\u003cstrong\u003eright\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9558913/v1/1659305fbd0d9269bf6dfa04.png"},{"id":109123563,"identity":"4f5a60b9-3468-4fb0-be4e-4e64591e2f7b","added_by":"auto","created_at":"2026-05-12 18:00:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25376617,"visible":true,"origin":"","legend":"\u003cp\u003eLight microscopic\u003cstrong\u003e \u003c/strong\u003eexamination didnot show any histological changes in the brain tissue of the rats from groups 1 (control) and 2 (A), while a downwardly directed convex hemispherical lesion was observed from the surface to the deeper part of the cortex in rats in groups3 to 8 (B). Magnified photo confirming features such as tissue vacuolation, swelling, and microbleeding (C).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9558913/v1/a5eb79ae9a8e30840338bde9.png"},{"id":109205134,"identity":"ee0a621c-e22a-4b74-b3b7-5cae126b5b8e","added_by":"auto","created_at":"2026-05-13 15:03:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1047316,"visible":true,"origin":"","legend":"\u003cp\u003eA comparison of lesion depth between rats stimulated with thesame total stimulation charge but different stimulation energiesin rats in groups 3 and 8 (\u003cstrong\u003eleft\u003c/strong\u003e) and stimulated with the same total stimulation energy but different stimulation chargesin rats in groups 3 and 7 (\u003cstrong\u003eright)\u003c/strong\u003e. Although the differences did not reach statistical significance, lesion depth was consistently greater in the higher-energy condition, whereas no difference was observed when total energy was equivalent (p = 0.25 and p = 1.0, respectively). Error bars represent ± standard deviation.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9558913/v1/e901ae63d6c3e5a414faa9ba.png"},{"id":109207286,"identity":"a0164567-450f-4a23-ad13-dfcce673da54","added_by":"auto","created_at":"2026-05-13 15:19:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":50096391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9558913/v1/b46c696f-bd45-4951-98ff-ef0f48809dd4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Energy-Dependent Cortical Injury Thresholds in High-Frequency Transcortical Electrical Stimulation: A Biophysical Study in a Rat Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntraoperative motor evoked potential (MEP) monitoring with transcortical stimulation is widely used to preserve motor pathways during brain surgery (Taniguchi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Krieg et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ichikawa et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The technique relies on brief trains of high-frequency pulses (typically several hundred Hz) delivered at intensities often exceeding those used in chronic cortical stimulation paradigms (Pechstein et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Oinuma et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Despite the widespread clinical use of this stimulation regime, the biophysical determinants of cortical tissue injury under such conditions remain poorly defined.\u003c/p\u003e \u003cp\u003eTraditional safety criteria for cortical stimulation, including Shannon\u0026rsquo;s model, emphasize charge density per phase and total charge density as primary predictors of neural injury (Shannon \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Cogan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gordon et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; MacDonald \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, these criteria were derived from chronic, low-frequency paradigms in which stimulation was delivered continuously over hours to days (Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Harnack et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The brief, high-frequency, repetitive trains used for intraoperative MEP differ fundamentally from these conditions in their temporal pattern of energy delivery and in the instantaneous power dissipated in tissue. Whether charge-based safety criteria adequately capture the biophysical relationship between stimulation parameters and the resulting volumetric tissue damage in this regime remains an open question.\u003c/p\u003e \u003cp\u003eIn the present study, we provide a mechanistic framework by evaluating the relative contributions of electrical charge (Q\u0026thinsp;=\u0026thinsp;I \u0026times; t) and stimulation energy (W \u0026prop; I\u0026sup2;t) to the depth of cortical lesions in a rat model under MEP-relevant high-frequency stimulation. By experimentally dissociating these two parameters\u0026mdash;comparing groups with matched total charge but different total energy, and vice versa\u0026mdash;we sought to identify the primary biophysical driver of tissue injury and to clarify the underlying physical mechanisms, including resistive heating and anodic electrolysis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThirty-two 8- to 12-week-old male Sprague-Dawley rats (weight 350\u0026ndash;450 grams) were used for the experiments. The rats were provided by the vivarium of Shinshu University School of Medicine. The animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by Shinshu University's Ethical Review Board. The rat experiment was approved by the Ethical Committee of Shinshu University (approval no. 200024). All procedures were designed in accordance with the 3R principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and to reduce suffering.\u003c/p\u003e \u003cp\u003eAfter induction of general anesthesia with intraperitoneal pentobarbital (60 mg/kg), all efforts were made to minimize pain and distress. The head was fixed to a rat head-fixation device. Bilateral frontal craniectomies with a diameter of 10 mm were performed, and the dura mater was exposed. The bilateral dura mater was incised circumferentially to expose the cerebral cortex. The arachnoid membrane and the cerebral cortex were kept intact. A pair of platinum electrodes with a diameter of 2 mm (Unique Medical Co. Ltd, Japan) were placed on the cerebral cortex bilaterally (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnodal monopolar high-frequency monophasic stimulation, which is a standard MEP stimulation method (Szel\u0026eacute;nyi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), was used to deliver electrical stimulation to the cerebral cortex. The placement of the stimulating electrode ensured that stimulation was delivered through the uninjured cortex. Neuropack Σ\u0026reg; (Nihon Kohden, Japan) with a constant-current stimulator was used as a stimulation machine. A five-pulse train stimulation with a pulse frequency of 500 Hz, an interstimulation interval (ISI) of 2 ms, a current of 20 mA or 50 mA, a duration of 0.2 ms or 0.05 ms, an interval between each stimulation session of 1s or 10s, and stimulus repetition of 0 (control), 10x, 100x, or 625x was delivered. This stimulation protocol resulted in 8 groups of rats (4 rats per group) with various combinations of stimulation parameters (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStimulation parameter for each group of the experiments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRats group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurrent (mA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuration (ms)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInterstimulation interval\u003c/p\u003e \u003cp\u003e(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStimulation number\u003c/p\u003e \u003cp\u003e(n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal charge (\u0026micro;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRelative\u003c/p\u003e \u003cp\u003eenergy\u003c/p\u003e \u003cp\u003e(I\u003csup\u003e2\u003c/sup\u003et, arbitrary units)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean lesion depth (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e209\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e157\u0026thinsp;\u0026plusmn;\u0026thinsp;141\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e183\u0026thinsp;\u0026plusmn;\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e211\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e125\u0026thinsp;\u0026plusmn;\u0026thinsp;101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEight experimental groups were designed to evaluate the following comparisons: (1) the effect of stimulation number (groups 1\u0026ndash;3 at 50 mA, and groups 4 and 7 at 20 mA); (2) the effect of stimulation current (groups 3 vs. 4, 50 mA vs. 20 mA, 100 repetitions); (3) the effect of stimulation duration (groups 3 vs. 5 at 50 mA, and groups 4 vs. 8 at 20 mA); (4) the effect of interstimulation interval (groups 3 vs. 6, 1 s vs. 10 s); (5) same total charge with different total energy (groups 3 vs. 8); (6) same total energy with different total charge (groups 3 vs. 7); and (7) the overall association between total charge or energy and lesion depth across all groups. Full stimulation parameters for each group are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTotal stimulation charge (Q) was defined as Q\u0026thinsp;=\u0026thinsp;I \u0026times; t, where I is the stimulation current, and t is the total stimulation time (pulse duration \u0026times; number of stimulations). Stimulation energy was expressed as proportional to I\u0026sup2;t (W \u0026prop; I\u0026sup2;t), assuming relatively constant tissue resistance across experimental conditions; accordingly, relative differences in I\u0026sup2;t were used as a surrogate for energy comparison across groups.\u003c/p\u003e \u003cp\u003eThe platinum electrodes were removed immediately after stimulation. Animals were humanely euthanized by an overdose of pentobarbital (150 mg/kg, intraperitoneally) in accordance with institutional and international guidelines. Whole-body perfusion fixation with 9% formaldehyde was performed, and then the removed brain was formalin-fixed. After appropriate fixation, the brain was vertically and coronally sectioned at the center position of the anodal electrode and then paraffin-fixed. A brain preparation was created with a thickness of 3.0 \u0026micro;m from the stimulated brain surface and stained with hematoxylin and eosin. Under an optical microscope, the maximum depth of the lesion in the cerebral cortex of the anodal stimulation was measured.\u003c/p\u003e \u003cp\u003eThe depth of the cerebral cortex lesion between the groups of rats was compared using the Mann-Whitney and Kruskal-Wallis tests. The correlation between stimulation charge and energy with the lesion depth was statistically analyzed with multiple linear regression. The p-value was considered significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analysis was performed using SPSS version 21.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTranscortical stimulation was delivered to all groups except the control (group 1) according to the research protocol. The electrode impedance was approximately 1 kΩ. The brain tissues under the anodal electrode were observed under an optical microscope. There were no histological changes found in rats in groups 1 (control) and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A downwardly directed convex hemispherical lesion was observed from the surface of the cortex to a deeper part of the cortex, consistent with the part of the electrode in contact with the disc plane in the rats in groups 3\u0026ndash;8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Tissue vacuolation, swelling, and microbleeding were confirmed inside the lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lesion depth, which was the distance between the cortical surface and the deepest part of the lesion, was measured. The lesion depth ranged from 0 \u0026micro;m to 296 \u0026micro;m, with an average of 122.8\u0026thinsp;\u0026plusmn;\u0026thinsp;101.3 \u0026micro;m. The average lesion depth for each group with their respective parameters of electrical stimulation is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The main findings are summarized below.\u003c/p\u003e \u003cp\u003eRegarding stimulation number, lesion depth increased significantly with greater repetition: groups 1 and 2 showed no lesion (0 \u0026micro;m), whereas group 3 reached 209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;m (p\u0026thinsp;=\u0026thinsp;0.005 for groups 1\u0026ndash;3), and group 7 reached 211\u0026thinsp;\u0026plusmn;\u0026thinsp;59 \u0026micro;m compared with 97\u0026thinsp;\u0026plusmn;\u0026thinsp;33 \u0026micro;m in group 4 (p\u0026thinsp;=\u0026thinsp;0.02). Higher stimulation current also produced significantly greater lesion depth: 209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;m (group 3, 50 mA) vs. 97\u0026thinsp;\u0026plusmn;\u0026thinsp;33 \u0026micro;m (group 4, 20 mA; p\u0026thinsp;=\u0026thinsp;0.04). All comparisons are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of different stimulation parameters toward lesion depth\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompared parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShared parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal charge (\u0026micro;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRat group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean lesion depth (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation number 1 (times)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC:50 mA, SD: 0.2 ms, SI: 1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation number 2 (times)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC:20 mA, SD: 0.2 ms, SI: 1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e625x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e211\u0026thinsp;\u0026plusmn;\u0026thinsp;59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation current (mA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSD: 0.2 ms, SN:100x, SI: 1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20 mA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50 mA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation duration 1 (ms)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC: 50 mA, SN: 100x, SI: 1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.05 ms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e157\u0026thinsp;\u0026plusmn;\u0026thinsp;141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.2 ms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation duration 2 (ms)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC: 20 mA, SN: 100x, SI: 1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.2 ms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5 ms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e125\u0026thinsp;\u0026plusmn;\u0026thinsp;101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation interval (s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC: 50 mA, SD: 0.2 ms, SN: 100x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e209\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e183\u0026thinsp;\u0026plusmn;\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eSC: stimulation current, SD: stimulation duration, SI: stimulation interval, SN: stimulation number\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn contrast, neither pulse duration nor interstimulation interval significantly affected lesion depth. Increasing duration from 0.05 to 0.2 ms (groups 5 vs. 3: 157\u0026thinsp;\u0026plusmn;\u0026thinsp;141 vs. 209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.56) and from 0.2 to 0.5 ms (groups 4 vs. 8: 97\u0026thinsp;\u0026plusmn;\u0026thinsp;33 vs. 125\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.25) produced numerically larger lesions, but the differences did not reach significance. Similarly, extending the interstimulation interval from 1 to 10 s (groups 3 vs. 6: 209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 vs. 183\u0026thinsp;\u0026plusmn;\u0026thinsp;36 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.09) showed a trend toward smaller lesions without reaching significance (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen total charge was held constant, but total energy differed (groups 3 vs. 8: 5,000 \u0026micro;C for both; relative energy I\u0026sup2;t: 250 vs. 100 [arbitrary units]), mean lesion depth was numerically greater in the higher-energy group (209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 vs. 125\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.25; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, left). Conversely, when total energy was equalized but total charge differed 2.5-fold (groups 3 vs. 7: 5,000 vs. 12,500 \u0026micro;C; relative energy I\u0026sup2;t: both 250 [arbitrary units]), mean lesion depths were virtually identical (209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 vs. 211\u0026thinsp;\u0026plusmn;\u0026thinsp;59 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;1.0; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, right). Multiple linear regression analysis across all groups yielded the equation: lesion depth\u0026thinsp;=\u0026thinsp;39.24\u0026thinsp;+\u0026thinsp;0.02 \u0026times; Q\u0026thinsp;+\u0026thinsp;0.61 \u0026times; W. Total stimulation energy was the only significant independent predictor (p\u0026thinsp;=\u0026thinsp;0.008), whereas total stimulation charge was not (p\u0026thinsp;=\u0026thinsp;0.677).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study examined the relative biophysical contributions of electrical charge and stimulation energy to cortical lesion depth under MEP-relevant high-frequency stimulation. Stimulation current and the number of stimulations were the parameters most consistently associated with greater lesion depth. Most importantly, controlled comparisons between groups with equivalent total charge but differing total energy demonstrated that parameters contributing to total stimulation energy (W \u0026prop; I\u0026sup2;t) play a greater role in determining lesion depth than total charge (Q\u0026thinsp;=\u0026thinsp;I \u0026times; t) alone, a pattern further supported by multiple regression in which total energy was the only significant independent predictor (p\u0026thinsp;=\u0026thinsp;0.008) while total charge was not (p\u0026thinsp;=\u0026thinsp;0.677). Although the small group sizes (n\u0026thinsp;=\u0026thinsp;4 per group) limited statistical power for detecting subtle effects of pulse duration or interstimulation interval, the consistency of the energy-dependent pattern across multiple controlled comparisons supports the validity of this mechanistic framework and identifies energy deposition as a dominant biophysical driver of cortical injury under these conditions.\u003c/p\u003e \u003cp\u003eElectrical tissue damage beneath a stimulating electrode is histologically characterized by neuronal vacuolation, edema, disruption of cytoarchitecture, vasodilation, and microhemorrhage (Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Multiple physicochemical mechanisms have been implicated, including gas evolution, local pH changes, resistive heating, and direct electrochemical effects on cellular membranes (MacDonald \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Berendson and Simonsson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Butterwick et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These factors accumulate with increasing stimulation intensity (Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Berendson and Simonsson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). At the anode in particular, oxidation reactions produce local acidification, which is considered a primary driver of tissue injury (Berendson and Simonsson \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The ohmic voltage drop across the electrode\u0026ndash;tissue interface contributes relatively little to tissue damage, as most of the applied charge is consumed in charging the electrical double layer rather than driving faradaic reactions. Collectively, these mechanisms converge through tissue electrolysis to produce the observed lesion pattern.\u003c/p\u003e \u003cp\u003ePrior work on chronic cortical stimulation has emphasized charge density per phase and total charge density as key determinants of neural injury, with McCreery et al. demonstrating a synergistic relationship between charge density and charge per pulse (Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Shannon's model similarly highlighted charge density as a primary predictor of damage thresholds (Cogan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gordon et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; MacDonald \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Shannon \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). However, those safety criteria were derived from chronic, low-frequency paradigms and may not directly translate to the brief, high-frequency, repetitive stimulation used for intraoperative MEP monitoring (Pechstein et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Oinuma et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Furthermore, inconsistencies in the relationship between charge density and neural damage across prior studies suggest that additional factors beyond charge alone may contribute to electrical injury during MEP-relevant stimulation.\u003c/p\u003e\n\u003ch3\u003eThe dominance of energy over charge in tissue injury\u003c/h3\u003e\n\u003cp\u003eThe present results suggest that, under MEP-relevant stimulation conditions, total energy may be a more informative predictor of lesion depth than total charge. In the key controlled comparison (Groups 3 vs. 8), both groups received identical total charge (5,000 \u0026micro;C), yet the group with higher total energy (relative energy I\u0026sup2;t: 250 vs. 100 [arbitrary units]) showed a numerically greater mean lesion depth (209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;m vs. 125\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.25). Conversely, when total energy was held constant while total charge differed by 2.5-fold (Groups 3 vs. 7: 5,000 \u0026micro;C vs. 12,500 \u0026micro;C), mean lesion depths were virtually identical (209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 \u0026micro;m vs. 211\u0026thinsp;\u0026plusmn;\u0026thinsp;59 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;1.0). Although neither comparison reached statistical significance\u0026mdash;likely reflecting the limited sample size\u0026mdash;the consistent directional pattern across both experiments supports the hypothesis that energy-related parameters drive lesion depth more than charge-related parameters under these conditions.\u003c/p\u003e\n\u003ch3\u003eBiophysical mechanisms: resistive heating and electrolysis\u003c/h3\u003e\n\u003cp\u003eA plausible mechanistic basis for this energy dependence lies in resistive heating. Because power dissipation in tissue scales with the square of current (P\u0026thinsp;=\u0026thinsp;I\u0026sup2;R), a higher stimulation current generates disproportionately more heat per unit time than a lower current delivering equivalent charge over a longer pulse duration. This I\u0026sup2; dependence of energy deposition may explain why stimulation current emerged as a significant predictor of lesion depth, while pulse duration\u0026mdash;despite influencing total charge\u0026mdash;did not. The pulse durations tested in the present study (0.05\u0026ndash;0.5 ms) span a range that overlaps with the chronaxie of cortical gray matter (0.2\u0026ndash;0.7 ms). Charge delivery near chronaxie is considered physiologically efficient for neural activation (Ranck \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Abalkhail et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but this same range may limit the independent contribution of pulse duration to electrochemical injury when current is held constant. The present results are consistent with this interpretation: in comparisons where total charge was equalized between groups but current differed (Groups 4 vs. 3 and Groups 4 vs. 8), lesion depth was consistently greater in the higher-current group, further suggesting that current magnitude\u0026mdash;and by extension, energy\u0026mdash;may be more relevant than pulse duration to the depth of cortical injury.\u003c/p\u003e \u003cp\u003eThe present findings may also help reconcile apparent discrepancies between our results and prior charge-density-based models (Cogan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yuen et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; McCreery et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Charge density tends to be highest at the electrode periphery, which may contribute predominantly to surface morphological damage rather than to the depth of injury measured here. Stimulation energy, by contrast, may better reflect the volumetric extent of tissue heating and electrolytic injury beneath the electrode. This distinction\u0026mdash;charge density as a predictor of surface morphological severity versus energy as a predictor of lesion depth or volume\u0026mdash;may help reconcile apparent discrepancies between prior histological studies and the present depth-based measurements.\u003c/p\u003e\n\u003ch3\u003eImplications for safer stimulation protocols\u003c/h3\u003e\n\u003cp\u003eThese findings carry several practical implications for the safe conduct of intraoperative MEP monitoring, bearing in mind their exploratory nature. When it is necessary to increase stimulation intensity\u0026mdash;for example, in patients with preoperative motor deficits, high stimulation thresholds, or monitoring of muscles with inherently high thresholds such as lower extremity musculature (Hardian et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u0026mdash;the present data suggest that increasing pulse duration rather than stimulation current may represent the safer strategy, as it increases total charge with a smaller proportional increase in energy. Keeping the pulse duration near the chronaxie of the target tissue would be expected to maintain stimulation efficiency while minimizing energy deposition (Abalkhail et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Second, the strong influence of stimulation number on lesion depth underscores the importance of threshold-based stimulation protocols that minimize unnecessary repetitions (Abboud et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Calancie et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hardian et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The threshold was defined in the present study as the lowest intensity yielding a reproducible MEP response with amplitude\u0026thinsp;\u0026ge;\u0026thinsp;20 \u0026micro;V and appropriate latency (Hardian et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Goto et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kanaya et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e), an approach that limits cumulative energy deposition while preserving monitoring sensitivity. When no MEP response is obtained, the stimulation number must be increased, though a certain additional margin may be acceptable to ensure reliable clinical monitoring.\u003c/p\u003e \u003cp\u003eIt is important to note that the stimulation parameters and electrode geometry used here are not directly representative of clinical practice. Standard intraoperative transcortical electrodes have a diameter of approximately 5 mm, whereas the 2 mm electrodes used in the present study are smaller. For equivalent stimulation current (20 mA), the charge density per phase beneath a 5 mm electrode is approximately 1/15 that of a 2 mm electrode, and if tissue change scales proportionally with energy, the effective damage would be expected to be substantially reduced (Goto et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kanaya et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Furthermore, in clinical practice, stimulation is typically delivered at intervals of several seconds to minutes rather than the 1-second interval used here, allowing greater thermal and electrochemical dissipation between trials. These differences suggest that tissue injury under typical clinical conditions would be substantially lower than that observed experimentally, consistent with the overall clinical safety record of transcortical MEP monitoring. Accordingly, the present findings should be interpreted as providing a mechanistic framework rather than a direct representation of clinical conditions, and caution should be exercised when extrapolating to the clinical setting (Taniguchi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Neuloh and Schramm \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Nevertheless, the present results serve as a reminder that apparently safe stimulation intensities may produce histological changes at the site of electrode contact, particularly under conditions of prolonged surgery or a high number of stimulation trials, and that minimizing total stimulation energy throughout the procedure remains prudent.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStudy limitations and exploratory nature\u003c/h2\u003e \u003cp\u003eSeveral limitations must be acknowledged. First, rat and human cortex differ in geometric organization and cellular density, and the tolerance of human cortex to electrical stimulation may not be identical to that of the rat (Hodge et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; DeFelipe \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Nevertheless, the fundamental cytoarchitecture and cellular constituents of the mammalian cerebral cortex are broadly conserved, supporting the relevance of the rat model for mechanistic investigation. Second, the present study measured lesion depth as a surrogate for injury severity. Whether the observed histological changes translate into functional motor deficits remains unknown and warrants dedicated investigation. Third, only a single electrode size was employed, precluding direct assessment of how electrode surface area modulates the relationship between energy, charge density, and injury depth. Variability in electrode contact area\u0026mdash;due to intraoperative handling or gas formation at the electrode\u0026ndash;tissue interface\u0026mdash;represents an additional source of uncertainty in both experimental and clinical settings. Fourth, the small group size (n\u0026thinsp;=\u0026thinsp;4 per group) substantially limits statistical power and generalizability. The non-significant p-values observed in several comparisons should therefore be interpreted with caution; they may reflect insufficient power to detect true differences rather than the absence of an effect. Replication with larger cohorts and a broader range of stimulation parameters is needed to confirm these observations. Finally, because brain tissue is electrically inhomogeneous and anisotropic (Astr\u0026ouml;m et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the simplified assumption of constant resistance used to calculate energy may introduce systematic error. In this study, stimulation energy was treated as a relative index proportional to I\u0026sup2;t, rather than an absolute quantity, because tissue resistance was not directly measured. Accordingly, the energy values reported here should be interpreted as relative comparisons between groups rather than true physical energy values. More refined models incorporating direct tissue impedance measurements would be required to validate this approach and strengthen future analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eUnder high-frequency transcortical stimulation conditions, the depth of cortical injury is more closely associated with total stimulation energy (W \u0026prop; I\u0026sup2;t) than with total electrical charge alone, consistent with biophysical mechanisms involving resistive heating and electrolytic processes at the electrode\u0026ndash;tissue interface. Stimulation current and the number of stimulations are the dominant determinants of energy deposition and, consequently, of lesion depth, whereas pulse duration and interstimulation interval play comparatively minor roles within the parameter ranges examined. These findings provide a mechanistic framework that may inform safer stimulation protocols: when stronger stimulation output is required, increasing pulse duration at constant current may be preferable to increasing current itself, as it raises total charge with a smaller proportional rise in energy. Replication in larger cohorts and with direct tissue impedance measurements is needed to establish quantitative thresholds across the parameter space.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals established by Shinshu University\u0026rsquo;s Ethical Review Board, and were approved by the Ethical Committee of Shinshu University (approval no. 200024). All procedures were designed in accordance with the 3R principles (Replacement, Reduction, and Refinement) to minimize the number of animals used and to reduce suffering. The reporting of this study conforms to the ARRIVE guidelines for animal pre-clinical research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003eNot applicable. This study did not involve human participants.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable. This study did not involve human participants.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRidzky Firmansyah Hardian: Conceptualization, Methodology, Investigation, Formal analysis, Writing \u0026ndash; original draft. Kunihiko Kodama: Conceptualization, Methodology, Investigation, Formal analysis, Writing \u0026ndash; original draft. Kohei Kanaya: Conceptualization, Methodology, Formal analysis, Writing \u0026ndash; review \u0026amp; editing, Supervision. Tetsuya Goto: Methodology, Writing \u0026ndash; review \u0026amp; editing, Supervision. Kazuhiro Hongo: Writing \u0026ndash; review \u0026amp; editing, Supervision. Tetsuyoshi Horiuchi: Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbalkhail TM, MacDonald DB, AlThubaiti I, AlOtaibi FA, Stigsby B, Mokeem AA et al (2017) Intraoperative direct cortical stimulation motor evoked potentials: Stimulus parameter recommendations based on rheobase and chronaxie. 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Neurosurgery 9:292\u0026ndash;299\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"transcortical stimulation, cortical lesion depth, biophysics, resistive heating, rat model","lastPublishedDoi":"10.21203/rs.3.rs-9558913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9558913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe biophysical determinants of cortical tissue injury during brief, high-frequency transcortical electrical stimulation remain incompletely understood. Traditional safety criteria derived from chronic, low-frequency paradigms emphasize total charge, but whether charge or stimulation energy is the primary predictor of injury under high-frequency conditions\u0026mdash;such as those used for intraoperative motor evoked potential (MEP) monitoring\u0026mdash;is unclear. Thirty-two Sprague-Dawley rats (8 groups of 4) received monophasic anodal transcortical pulse trains with varying current, pulse duration, repetition number, and interstimulation interval. Maximum cortical lesion depth was measured histologically, and the contributions of total charge (Q\u0026thinsp;=\u0026thinsp;I \u0026times; t) and relative stimulation energy (W \u0026prop; I\u0026sup2;t) were dissociated through controlled group comparisons and multiple linear regression. Higher stimulation current (p\u0026thinsp;=\u0026thinsp;0.04) and greater repetition number (p\u0026thinsp;=\u0026thinsp;0.005) significantly increased lesion depth. Multiple regression identified total stimulation energy as the only significant independent predictor (p\u0026thinsp;=\u0026thinsp;0.008), whereas total charge was not (p\u0026thinsp;=\u0026thinsp;0.677). With equivalent total charge, higher-energy stimulation tended to produce deeper lesions (209\u0026thinsp;\u0026plusmn;\u0026thinsp;67 vs. 125\u0026thinsp;\u0026plusmn;\u0026thinsp;101 \u0026micro;m, p\u0026thinsp;=\u0026thinsp;0.25); when total energy was equalized despite a 2.5-fold difference in total charge, lesion depths were nearly identical (p\u0026thinsp;=\u0026thinsp;1.0). Under high-frequency conditions, cortical injury depth is governed primarily by total energy deposition rather than total charge, consistent with biophysical mechanisms involving resistive heating and electrolytic processes at the electrode\u0026ndash;tissue interface. These findings provide a mechanistic framework for understanding electrical tissue injury under MEP-relevant stimulation regimes.\u003c/p\u003e","manuscriptTitle":"Energy-Dependent Cortical Injury Thresholds in High-Frequency Transcortical Electrical Stimulation: A Biophysical Study in a Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 18:00:31","doi":"10.21203/rs.3.rs-9558913/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"81d9ba7c-cbf8-4138-b6aa-d996e2c2547e","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-11T13:28:43+00:00","index":23,"fulltext":""},{"type":"reviewerAgreed","content":"147252604977866887188711532448878804874","date":"2026-05-09T18:02:08+00:00","index":22,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T06:11:17+00:00","index":21,"fulltext":""},{"type":"reviewerAgreed","content":"312111922023146744925807106037835867136","date":"2026-05-07T05:07:54+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"331475669467652283682171154352667047685","date":"2026-05-04T08:26:57+00:00","index":14,"fulltext":""},{"type":"reviewersInvited","content":"9","date":"2026-05-04T06:14:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T07:27:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T07:27:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental Brain Research","date":"2026-04-29T00:46:25+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T18:00:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 18:00:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9558913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9558913","identity":"rs-9558913","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}