Levobupivacaine induces seizures in Wistar rats through behavioral, electrophysiological, and pharmacological modulation

Levobupivacaine induces seizures in Wistar rats through behavioral, electrophysiological, and pharmacological modulation | 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 Levobupivacaine induces seizures in Wistar rats through behavioral, electrophysiological, and pharmacological modulation Axell Lins, Daniella Bastos Araújo, Luciana Eiró-Quirino, Clarissa Araújo Paz, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7973054/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Status epilepticus (SE) remains a life-threatening neurological emergency driven by prolonged, excessive neuronal discharges. At toxic concentrations, levobupivacaine (LBE), a potent local anesthetic, can provoke seizures and thus serves as a practical model to study seizure generation and therapeutic response. We established an LBE-induced seizure model in adult Wistar rats and combined behavioral scoring, hippocampal field recordings, and pharmacological intervention. Following intraperitoneal LBE (20 mg/kg), animals progressed reproducibly through four behavioral stages (mean latencies: 119.6 s, 154.8 s, 175.9 s, and 225.1 s), ending in tonic–clonic events complicated by respiratory failure. Electrophysiological traces showed alternating ictal and interictal epochs with high amplitude burst discharges and significant increases in spectral power across theta, beta, and gamma bands. Intravenous administration of diazepam and phenobarbital markedly suppressed ictal discharges; phenytoin and valproate yielded only partial attenuation. Collectively, the LBE model reproduces key behavioral and electrographic hallmarks of convulsive SE and provides a reproducible platform for probing convulsant neurotoxicity and for preclinical screening of anticonvulsant strategies. Levobupivacaine Seizure model Status epilepticus Electrophysiology Anticonvulsants Wistar rats Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Status epilepticus (SE) is a severe neurological emergency characterized by high mortality and significant underreporting (Ameli et al., 2021 ; Lattanzi et al., 2023 ). SE results from abnormal, excessive neuronal activity in the brain exceeding 2 Hz, and can present as generalized (bilateral involvement), focal, or bilateral independent (distinct electroencephalographic patterns across hemispheres) (Rosenthal, 2021 ; Beniczky et al., 2013 ). Clinically, SE manifests with absence seizures, impaired consciousness, motor abnormalities, or convulsions (Leitinger et al., 2016 ). These features occur during the ictal period, defined by electroencephalographic alterations, while the interictal period represents the interval between episodes, when neuronal discharges approximate baseline levels (Tedrus, 2024 ; Meletti et al., 2023 ). During ictal episodes, patients may exhibit tonic–clonic seizures or other clinical manifestations (Johnson et al., 2020). Continuous electroencephalography (EEG) monitoring is therefore essential to accurately distinguish ictal from interictal phases (Merli et al., 2022 ; Struck et al., 2017 ). Seizures are paroxysmal events caused by hypersynchronous and excessive neuronal discharges in focal or diffuse brain regions, leading to transient neurological dysfunction. Clinically, they present with loss of consciousness, muscle rigidity, and abrupt, uncontrolled skeletal movements. A diagnosis of epilepsy is established when two or more unprovoked or reflex seizures occur > 24 hours apart, or when recurrence risk exceeds 60% within 10 years (Bernard et al., 2014 ; Chauhan et al., 2022 ). The hippocampus, a structure involved in memory and neural plasticity, is particularly vulnerable to hypersynchronous discharges. Evidence indicates acute hippocampal injury in SE, with histopathological signs of neuronal necrosis linked to hypoperfusion and metabolic disturbances (Postnikova et al., 2022 ; Zhvania et al., 2025 ). Hippocampal damage is multifactorial, influenced by age at first insult, genetic predisposition, and autoimmune etiologies. Prolonged febrile seizures, for example, can induce functional and morphological changes culminating in persistent CA1 injury (Griflyuk et al., 2023 ). Levobupivacaine hydrochloride (LBE), the S(–)-enantiomer of bupivacaine, belongs to the amide class of local anesthetics. It was developed to reduce neuro- and cardiotoxicity compared with racemic bupivacaine, while maintaining high lipophilicity, potency, and long duration of action (Steverink et al., 2021 ; Yang et al., 2022 ; Jagan et al., 2024 ). Clinically, it is used for regional anesthesia and analgesia, including epidural, spinal, peripheral nerve blocks, local infiltration, postoperative pain, and continuous infusion for chronic pain (Naithani et al., 2025 ; Li et al., 2025 ). Mechanistically, levobupivacaine reversibly blocks voltage-gated sodium channels (α-subunit), preventing depolarization and action potential propagation, with stereoselective effects (Steverink et al., 2021 ; Chalkiadis et al., 2005 ; Bajwa & Kaur, 2013 ). Although safer than bupivacaine, levobupivacaine can cause mild neurological symptoms (e.g., tinnitus, blurred vision) at high doses, and in severe cases may progress to seizures, coma, and respiratory arrest. Cardiovascular adverse effects include bradycardia and arrhythmias (Abut et al., 2015 ; Bajwa & Kaur, 2013 ; Reddy et al., 2021 ; Uzun & İdin, 2025 ). Given this context, the role of antiseizure medications (ASMs) is of particular relevance, since understanding how crises are interrupted provides insight into the mechanisms underlying SE. Among the most widely used ASMs, diazepam and phenobarbital enhance inhibitory neurotransmission through GABA-A receptors, increasing chloride influx and reducing neuronal excitability (Fritschy, 2008 ; Althaus et al., 2020 ; Foitzick et al., 2020 ; Zhang et al., 2022 ; Jones et al., 2022 ; Men & Wang, 2023 ; Li et al., 2025 ). Phenytoin acts by blocking voltage-gated sodium channels in their inactivated state, whereas sodium valproate exerts a broader mechanism that includes elevating GABA levels, modulating ion channels, and inhibiting histone deacetylases (Samanta, 2021 ; Goldberg, 2021 ; Bousman et al., 2021 ; Mishra et al., 2021 ; Kumar, 2022 ; Anguissola et al., 2023 ; Shakerdi & Ryan, 2023 ; Milosavljevic et al., 2024 ; Kong et al., 2024 ; Gziut & Thanacoody, 2025 ). Together, these drugs illustrate the diversity of therapeutic strategies employed to counteract cerebral hyperexcitability. Therefore, considering that local anesthetics administered in high doses or by continuous infusion can trigger seizure episodes, this study aimed to develop a levobupivacaine-induced seizure model and to analyze its behavioral and electrophysiological patterns, as well as the response to pharmacological control. 2. Materials and Methods 2.1 Animals A total of 72 adult male Wistar rats (200 ± 20 g; 10 weeks old) were obtained from the Central Animal Facility of the Federal University of Pará. The animals were housed in standard white cages (48 × 38 × 21 cm) under controlled environmental conditions (22 ± 2°C; 12/12 h light/dark cycle), with free access to food and water. All experimental procedures were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Animal Ethics Committee of the Federal University of Pará (CEUA No. 6135040822). All necessary measures were taken to minimize animal suffering and distress. 2.2. Drugs Ketamine was purchased from König and xylazine from Vallée, while levobupivacaine (LBE) was obtained from Cristália Laboratory. Anticonvulsant compounds were sourced from different suppliers: phenobarbital (PHB) from Aventis Pharma, phenytoin (PHT) and diazepam (DZP) from União Química, and injectable sodium valproate (Depacon) from Abbott Laboratories do Brasil Ltda. (São Paulo, Brazil). 2.3. Experimental Design The study comprised three experiments. In Experiment 1, seizure-related behavior was described in the LBE group (n = 9), which received a single intraperitoneal (i.p.) dose of 20 mg/kg. Behavioral activity was assessed by measuring the latency to onset of convulsive behavior (in seconds). In Experiment 2, rats were randomly assigned to receive either vehicle (control) or levobupivacaine (20 mg/kg i.p.; n = 9). Electrophysiological recordings were obtained from the CA1 region of the hippocampus to characterize the recorded traces. In Experiment 3, animals were treated with four anticonvulsant drugs (n = 9 per group), following the protocol described by Hamoy et al. ( 2018 ): (a) diazepam (DZP, 10 mg/kg i.v.), (b) phenobarbital (PHB, 10 mg/kg i.v.), (c) phenytoin (PHT, 10 mg/kg i.v.), and (d) sodium valproate (VPA, 10 mg/kg i.v.). In all groups, hippocampal recordings were performed. All drugs were administered via intravenous injection into the lateral tail vein. The vehicle group received 0.9% saline solution in a volume (ml) equivalent to body weight (kg). 2.4. Description of Seizure-Related Behavior Seizure-related behavior was observed following LBE administration. Seizure latency was recorded, and behavioral modifications were classified into four rapidly evolving, clinically identifiable stages: (a) bristling of whiskers, akinesia, and motionless staring; (b) generalized tremor without loss of the righting reflex; (c) generalized clonus with loss of the righting reflex; (d) clonic spasms with labored breathing, followed by respiratory arrest. 2.5. Hippocampal Recordings and Data Analysis Hippocampal recordings were obtained using the procedures described by Quirino et al. (2024). Animals were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.) and xylazine hydrochloride (5 mg/kg, i.p.) and, after the abolition of the interdigital reflex, placed in a stereotaxic apparatus. The skull was exposed, and stainless-steel electrodes (0.03 mm in diameter, 3.4 mm in length), insulated with Teflon except for a 0.5 mm exposed tip, were implanted for signal acquisition. Electrodes were positioned in the CA1 region of the hippocampus according to the following stereotaxic coordinates: bregma − 3.00 mm, 1.6 mm lateral, and 3.40 mm dorsoventral. Five days after surgery, animals were treated with LBE, and electrodes were connected to a high-gain amplifier (Fig. 1 A and B). Recordings were performed following a standardized protocol. Animals were gently restrained for 10 minutes to allow adaptation and to minimize artifacts. Baseline hippocampal activity was recorded for 10 minutes and used as control in the analyses. Immediately after LBE administration, hippocampal activity was recorded for 10 minutes. Since LBE-induced seizures progressed rapidly to respiratory arrest, animals were euthanized at this stage to prevent further suffering. For evaluation of LBE-induced seizures controlled by anticonvulsants, animals were treated following a similar protocol. After receiving one of the anticonvulsant drugs (DZP, PHB, PHT, or VPA) via intravenous injection into the lateral tail vein, immediately after LBE administration (i.p.), hippocampal recordings were performed for 10 minutes. 2.6 Data Analysis Recordings were obtained using a differential amplifier with high input impedance in AC mode (Grass Technologies, Model P511), adjusted with a 0.3 Hz–3 kHz band-pass filter. Signals were monitored with an oscilloscope (Protek, Model 6510) and continuously digitized at a rate of 1 kHz by a computer equipped with a data acquisition board (National Instruments, Austin, TX). Seizure characterization was performed using Python programming language (version 2.7) and the Signal® 3.0 software. Signal acquisition analyses were conducted in the frequency range up to 40 Hz, subdivided into theta (4–8 Hz), beta (12–28 Hz), and gamma (28–40 Hz) bands, to interpret the dynamics of seizure development (Hamoy et al., 2018 ). 2.7 Euthanasia After completion of the experiments, animals were euthanized with a lethal intraperitoneal injection of ketamine (300 mg/kg) combined with xylazine hydrochloride (30 mg/kg, i.p.) and diazepam (10 mg/kg, i.p.). This procedure was necessary to prevent animal suffering. 2.8 Statistical Analysis Results were subjected to descriptive statistics, expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test. Data were analyzed using GraphPad Prism, version 8 (GraphPad Software Inc.). Statistical significance was considered at p < 0.05, * p < 0.01, and ** p < 0.001. 3. Results 3.1. Seizure-Related Behavior Induced by LBE Seizure-related behavior was observed after intraperitoneal injection of LBE at a dose of 20 mg/kg. Animals exhibited continuous and progressive seizure stages. The mean latency for the first behavioral change—whisker bristling, akinesia, and motionless posture—was 119.6 ± 10.51 s, followed by generalized tremor without loss of the righting reflex (stage 2) at 154.8 ± 12.29 s. Generalized clonic seizures with transient loss of the righting reflex (stage 3) occurred at 175.9 ± 15.44 s, and tonic–clonic seizures with labored breathing, followed by respiratory arrest (stage 4), were observed at 225.1 ± 28.46 s (Table 1 ). Table 1 Mean latency for the onset of behaviors observed after levobupivacaine treatment in rats. Behavior Bristling of whiskers, akinesia and motionless staring Generalized tremor without loss of righting reflex Generalized clonus with transient loss of righting reflex Tonic-clonic seizure with labored breathing, followed by respiratory arrest Latency (s) (average and standard deviation) 119.6 ± 10.51 (s) 154.8 ± 12,29 (s) 175.9 ± 15.44 (s) 225.1 ± 28.46 (S) Recordings from the control group showed low-amplitude signals averaging 0.1 mV, with the highest energy concentrated below 10 Hz (Fig. 2 A). In contrast, the LBE group exhibited hippocampal alterations with cyclic peaks exceeding 0.4 mV, characteristic of burst potentials and indicative of seizure activity (Fig. 2 B, left). The LBE group displayed a characteristic convulsive pattern, with distinct amplitude variations during ictal and interictal periods (Fig. 2 B, center). During ictal periods, characteristic graphoelements of seizure syndromes (spike–wave and polyspike complexes) were identified in the hippocampus. The spectrogram demonstrated seizure power and the rapid progression to respiratory arrest, which reduced spectrogram power (Fig. 2 B, right). During seizures, analysis of ictal and interictal periods induced by LBE injection demonstrated power fluctuations. The power spectral density (PSD) plot revealed higher power across all frequency bands during the ictal period compared with both the LBE baseline and the interictal recording. The largest power during the ictal state occurred in the theta (4–8 Hz), beta (12–28 Hz), and gamma (28–40 Hz) ranges (Fig. 3 A). Significant between-group differences were observed across the frequency range up to 40 Hz. The control group showed a mean power of 0.259 ± 0.0275 mV²/Hz × 10⁻³, similar to the interictal period (p = 0.972). The ictal period exhibited higher total spectral power (3.307 ± 1.044 mV²/Hz × 10⁻³) than all other conditions (Fig. 3 B). For theta oscillations, the control group (0.0802 ± 0.0109 mV²/Hz × 10⁻³) did not differ from the interictal period (p = 0.998) but was lower than the remaining groups. The ictal period averaged 1.344 ± 0.278 mV²/Hz × 10⁻³, exceeding all others (Fig. 3 C). For beta oscillations, the control group had a mean of 0.03797 ± 0.00578 mV²/Hz × 10⁻³, comparable to the interictal group (p = 0.916) yet lower than the others. Mean power during the ictal period (1.09 ± 0.166 mV²/Hz × 10⁻³) was higher than in all other groups (Fig. 4 D). For gamma oscillations, the control group (0.0080 ± 0.0014 mV²/Hz × 10⁻³) was similar to the interictal group (p = 0.983) but lower than the remaining groups. Mean power during the ictal period (0.228 ± 0.045 mV²/Hz × 10⁻³) exceeded all other groups (Fig. 4 E). Anticonvulsants were tested to determine whether they could reduce or abolish alterations in linear power recorded in the hippocampus by analyzing oscillatory power up to 40 Hz and in the beta range (12–28 Hz). For this purpose, following intraperitoneal administration of levobupivacaine, PHT, PHB, DZP, and VPA were administered. Hippocampal recordings showed that the lowest amplitude was observed in the DZP group, while DZP and PHB provided the most effective seizure control (Fig. 4 A–D). To evaluate anticonvulsant activity, mean linear power was compared across groups. The control group showed 0.259 ± 0.0275 mV²/Hz × 10⁻³, which was similar to the LBE + PHB group (p = 0.996) and the LBE + DZP group (p = 0.999). The group treated with LBE alone (2.08 ± 0.751 mV²/Hz × 10⁻³) exhibited higher values than all other groups. The LBE + PHT group (1.005 ± 0.461 mV²/Hz × 10⁻³) was similar to the LBE + VPA group (p = 0.963). The LBE + PHB group (0.345 ± 0.081 mV²/Hz × 10⁻³) was comparable to the LBE + DZP group (p = 0.999) and to the LBE + VPA group (p = 0.0508) (Fig. 4 E). To further assess anticonvulsant activity, mean linear power in the beta range was compared across groups. The control group (0.0379 ± 0.00578 mV²/Hz × 10⁻³) was similar to the LBE + PHB group (p = 0.989) and the LBE + DZP group (p = 0.999). The group treated with LBE alone (0.718 ± 0.114 mV²/Hz × 10⁻³) exhibited higher values than all other groups. The LBE + PHT group (0.061 ± 0.0117 mV²/Hz × 10⁻³) was like the LBE + VPA group (p = 0.996) (Fig. 4 F). 4. Discussion The levobupivacaine group exhibited a convulsive state with amplitude oscillations, alternating between ictal and interictal periods, similar to the experimental model of lidocaine-induced seizures in rats standardized by Santos et al. ( 2020 ). That study demonstrated a behavioral and electroencephalographic progression that also differentiated ictal from interictal phases. In this context, lidocaine at toxic levels (60 mg/kg, i.p.) induced seizures defined by behavioral stages ranging from initial akinesia to generalized tonic–clonic seizures. In the EEG, epileptiform alterations were observed, including high-amplitude, high-frequency discharges during the ictal phase, interspersed with periods of interictal activity. A comparable pattern was observed in the LBE group, where ictal activity mirrored that described in human seizure conditions. Similarly, Azevedo et al. ( 2022 ) reported in a rat model of caffeine-induced seizures that beta-band activity (12–28 Hz) increased with high-frequency oscillations during the ictal state, corroborating our findings. In our study, ictal and interictal phases were clearly distinguished, with repeated sharp waves and high-amplitude discharges, supporting the concept that both LBE and caffeine generate electrodynamic seizure patterns with comparable detectability. This validates their use as models for studying electrophysiological seizure markers. Such evidence reinforces that convulsant mechanisms, whether via adenosine antagonism with caffeine or sodium channel blockade with LBE, can be reliably detected in animal models. This observation is in line with Hamoy et al. ( 2018 ), in which intraperitoneal infusion of cunaniol elicited interictal activity characterized by interspersed spikes preceding ictal episodes, correlating directly with behavioral patterns. Comparatively, in the acute seizure model induced by pentylenetetrazol (PTZ), (de Araújo et al., 2024 ) demonstrated that gamma oscillation power undergoes significant alterations during PTZ-induced seizures, with a reduction in gamma power noted during the postictal period. In contrast, our study revealed that gamma-band power increased markedly during ictal episodes, indicating intense neural activity typical of seizure onset. During interictal and control phases, gamma power remained low and stable, clearly distinguishing physiological states (control/interictal) from the pathological state (ictal) in terms of high-frequency activity. Supporting this, Zou et al. ( 2024 ) showed that oral administration of ciprofloxacin (CPC) for 14 days at therapeutic doses in rats increased susceptibility to seizures when PTZ was administered at higher doses. Interestingly, cortical CPC accumulation was not elevated, suggesting that seizure sensitization resulted from interference with GABA-A receptors and enhanced NMDA activity. Given the similarity between LBE- and PTZ-induced discharges, CPC sensitization may increase the risk of severe seizure episodes. Neurotoxicity induced by LBE, particularly under continuous infusion or overdose, is characterized by multiple spike-and-slow-wave complexes, as demonstrated in our results. Based on this, Noji et al. ( 2025 ) reported a cumulative threshold dose of 28.63 mg/kg for LBE, which is considered relatively low compared with other agents. In contrast, bupivacaine displays a more specific toxicity profile. Lai (2022) reported activation of the NLRP3 inflammasome, increased caspase-1 activity, and GSDMD-N expression. Additionally, Yilmaz, Tepe, and Uludağ (2023) observed in rats that renal impairment alters pharmacokinetics, enhancing central nervous system toxicity due to prolonged exposure. This was associated with activation of TRPM2 (Transient Receptor Potential Melastatin 2) triggered by oxidative stress and mitochondrial dysfunction, along with increased Reelin expression leading to synaptic dysfunction and impaired neural plasticity. Collectively, these findings demonstrate that bupivacaine exhibits higher toxicity levels than LBE. During the onset of behavioral alterations, a clear temporal progression of seizure stages was identified: 119.6 ± 10.51 s – Stage 1; 154.8 ± 12.29 s – Stage 2; 175.9 ± 15.44 s – Stage 3; and subsequently, tonic–clonic seizures with labored breathing and respiratory arrest (Stage 4). This corroborates Noji et al. ( 2025 ), who reported similar transitions from Stages 1–3 accompanied by the onset of ictal discharges (≥ 100 mV in the EEG at ≥ 1 Hz). Similarly, Ohmura et al. ( 2001 ) and Cheng et al. ( 2016 ) noted the predominance of Stages 2 and 3 (tremors and clonic seizures with loss of posture), as well as respiratory and circulatory collapse during Stage 4. In this context, attempts to increase the seizure threshold have included pharmacological interventions. Tanaka et al. ( 2005 ) demonstrated that dexmedetomidine prolonged the time and dose required for seizure onset, although progressive stages (prodromal signs, tremors, clonic, and tonic–clonic seizures) were still observed, consistent with our findings. Likewise, Oda and Ikeda ( 2013 ) used lipid emulsion with the same objective and successfully delayed seizure onset. More specifically, seizure initiation was postponed; however, progression still predominated from Stage 2 onward. Conclusion Therefore, the findings demonstrate that levobupivacaine, compared to other seizure models, consistently reproduces behavioral and electroencephalographic patterns characteristic of epileptic seizures, clearly distinguishing the ictal and interictal phases, with specific alterations in the fast bands. Furthermore, it was observed to have a relatively low toxic threshold, with neurotoxicity associated with multiple mechanisms, including mitochondrial dysfunction, inflammatory activation, and oxidative stress, making it more harmful than bupivacaine in certain contexts. It was also observed that anticonvulsants showed differentiated responses, with diazepam and phenobarbital demonstrating the greatest efficacy, significantly abolishing epileptiform discharges. In contrast, phenytoin and valproate showed only partial effect, attenuating but not completely reversing ictal activity, thus being less effective and, in some cases, refractory to immediate seizure control. Declarations Author contributions Conceived and designed the experiments: A.L. and M.H. Performed the experiments: A.L, D.B.A., L.E.Q., C.A.P., T.S.R. and M.H. Writing-original draft and editing: all authors. Financial support and administrative support: M.H. All authors have read and agreed to the published version of the manuscript. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Coordination for the Improvement of Higher Education Personnel. Acknowledgements Thanks to the Coordination for the Improvement of Higher Education Personnel (Brazilian CAPES), Amazon Foundation for Support of Studies and Research of the State of Pará (FAPESPA), post-graduation in pharmacology and biochemistry of Federal University of Pará (PPGFARMABIO). The authors also thank the students and staff of the Laboratory of Toxicology of Natural Products (UFPA – Belém) for developing the techniques that allowed the evaluation of electrophysiological activity. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Clinical trial number The study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable. Consent to participate No human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable. Consent publish All authors have read and approved the final version of the manuscript and consent to its publication. Ethical Approval and Accordance All procedures involving animals were approved by the Animal Use Ethics Committee of the Federal University of Pará (CEUA/UFPA, Protocol No. 6135040822). Experimental research on vertebrates was conducted in accordance with institutional, national, and international ethical guidelines for animal research and complies with the principles of the Basel Declaration (https://animalresearchtomorrow.org/en). Consent to Participate No human participants were involved. The study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement; therefore, this item is not applicable. DAS statement request All data generated or analyzed during this study are available in the public repository at the following link: https://drive.google.com/file/d/1IzKv17hS0-QHNk97laZWnfFttXH_xXPk/view?usp=sharing. The datasets support the findings of this study and include the original electrophysiological and behavioral data. Conflict of interest 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. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Gut microbiota and serum metabolomic alterations in modulating the impact of fecal microbiota transplantation on ciprofloxacin-induced seizure susceptibility. Frontiers in microbiology, 15, 1403892. https://doi.org/10.3389/fmicb.2024.1403892 Additional Declarations No competing interests reported. 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03:33:39","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143380,"visible":true,"origin":"","legend":"","description":"","filename":"952496474f5d4bbaa671f6ffdbce17ee1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/5c29972742a7f46ffe4e45a8.xml"},{"id":96134659,"identity":"458cebd1-abe7-4de0-9671-e48dc5c70eca","added_by":"auto","created_at":"2025-11-18 03:33:39","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154930,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/5cf0828a911ba96e5d391aeb.html"},{"id":96134621,"identity":"f2e4cfc4-9997-424f-9add-49529b965333","added_by":"auto","created_at":"2025-11-18 03:33:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":408049,"visible":true,"origin":"","legend":"\u003cp\u003eElectrode used in the experiment, made of stainless steel, 0.03 mm in diameter (A). Electrode placement for recording in the CA1 region of the rat hippocampus (B). Image modified from \u003cem\u003ePaxinos and Franklin's the mouse brain in stereotaxic coordinates.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/83d6c53a0351846e2dd399d6.png"},{"id":96248764,"identity":"01f13668-14b0-49d8-b4f4-967a487f75e0","added_by":"auto","created_at":"2025-11-19 07:29:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":228072,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal recordings showing field potential activity captured by the electrode over 10 minutes (left), amplification of recordings for graphoelement evaluation over 10 seconds (center), and spectrogram of power distribution over 10 minutes (right). Data are presented for the following groups: control group (A) and LBE-treated group showing ictal and interictal periods (B).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/c694ac28ebba060825a64025.png"},{"id":96134622,"identity":"e8ebe0f7-9cf7-4296-aaeb-94fe678ff596","added_by":"auto","created_at":"2025-11-18 03:33:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":238429,"visible":true,"origin":"","legend":"\u003cp\u003eThe graph illustrates the power spectral density (PSD) for mean power values analyzed up to 40 Hz during hippocampal recordings in the LBE-treated group and during ictal and interictal periods induced by LBE seizures. The panels show the linear power distribution during LBE-induced seizures up to 40 Hz (C), the linear power distribution of theta oscillations (4–8 Hz) (D), the linear power distribution of beta oscillations (12–28 Hz) (E), and the linear power distribution of gamma oscillations (28–40 Hz) (F). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 9).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/3df367e16c764de38ef26fba.png"},{"id":96134624,"identity":"4ca0733e-4605-4418-a40c-a59045e8444c","added_by":"auto","created_at":"2025-11-18 03:33:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234485,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal recordings from rats treated with LBE followed by intravenous administration of anticonvulsants, recorded for 600 s (left), and power spectrograms for the following groups: LBE + PHT (A), LBE + PHB (B), LBE + DZP (C), and LBE + VPA (D). Linear frequency distribution of total power during control of LBE-induced seizures (E) and control of LBE-induced seizures in the beta oscillation range (F). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 9).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/d8d35f04cb59dc2ef8d19f50.png"},{"id":96257053,"identity":"42fa79b3-ed6c-4c8c-9145-6b083881bc42","added_by":"auto","created_at":"2025-11-19 07:51:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1802122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7973054/v1/972bf90e-0ec5-43e2-8985-35f59d16278f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Levobupivacaine induces seizures in Wistar rats through behavioral, electrophysiological, and pharmacological modulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStatus epilepticus (SE) is a severe neurological emergency characterized by high mortality and significant underreporting (Ameli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lattanzi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). SE results from abnormal, excessive neuronal activity in the brain exceeding 2 Hz, and can present as generalized (bilateral involvement), focal, or bilateral independent (distinct electroencephalographic patterns across hemispheres) (Rosenthal, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Beniczky et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eClinically, SE manifests with absence seizures, impaired consciousness, motor abnormalities, or convulsions (Leitinger et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These features occur during the ictal period, defined by electroencephalographic alterations, while the interictal period represents the interval between episodes, when neuronal discharges approximate baseline levels (Tedrus, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Meletti et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDuring ictal episodes, patients may exhibit tonic\u0026ndash;clonic seizures or other clinical manifestations (Johnson et al., 2020). Continuous electroencephalography (EEG) monitoring is therefore essential to accurately distinguish ictal from interictal phases (Merli et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Struck et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeizures are paroxysmal events caused by hypersynchronous and excessive neuronal discharges in focal or diffuse brain regions, leading to transient neurological dysfunction. Clinically, they present with loss of consciousness, muscle rigidity, and abrupt, uncontrolled skeletal movements. A diagnosis of epilepsy is established when two or more unprovoked or reflex seizures occur\u0026thinsp;\u0026gt;\u0026thinsp;24 hours apart, or when recurrence risk exceeds 60% within 10 years (Bernard et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chauhan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe hippocampus, a structure involved in memory and neural plasticity, is particularly vulnerable to hypersynchronous discharges. Evidence indicates acute hippocampal injury in SE, with histopathological signs of neuronal necrosis linked to hypoperfusion and metabolic disturbances (Postnikova et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhvania et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Hippocampal damage is multifactorial, influenced by age at first insult, genetic predisposition, and autoimmune etiologies. Prolonged febrile seizures, for example, can induce functional and morphological changes culminating in persistent CA1 injury (Griflyuk et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLevobupivacaine hydrochloride (LBE), the S(\u0026ndash;)-enantiomer of bupivacaine, belongs to the amide class of local anesthetics. It was developed to reduce neuro- and cardiotoxicity compared with racemic bupivacaine, while maintaining high lipophilicity, potency, and long duration of action (Steverink et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jagan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Clinically, it is used for regional anesthesia and analgesia, including epidural, spinal, peripheral nerve blocks, local infiltration, postoperative pain, and continuous infusion for chronic pain (Naithani et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Mechanistically, levobupivacaine reversibly blocks voltage-gated sodium channels (α-subunit), preventing depolarization and action potential propagation, with stereoselective effects (Steverink et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chalkiadis et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bajwa \u0026amp; Kaur, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough safer than bupivacaine, levobupivacaine can cause mild neurological symptoms (e.g., tinnitus, blurred vision) at high doses, and in severe cases may progress to seizures, coma, and respiratory arrest. Cardiovascular adverse effects include bradycardia and arrhythmias (Abut et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bajwa \u0026amp; Kaur, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Reddy et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Uzun \u0026amp; İdin, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven this context, the role of antiseizure medications (ASMs) is of particular relevance, since understanding how crises are interrupted provides insight into the mechanisms underlying SE. Among the most widely used ASMs, diazepam and phenobarbital enhance inhibitory neurotransmission through GABA-A receptors, increasing chloride influx and reducing neuronal excitability (Fritschy, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Althaus et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Foitzick et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jones et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Men \u0026amp; Wang, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Phenytoin acts by blocking voltage-gated sodium channels in their inactivated state, whereas sodium valproate exerts a broader mechanism that includes elevating GABA levels, modulating ion channels, and inhibiting histone deacetylases (Samanta, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Goldberg, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bousman et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mishra et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Anguissola et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shakerdi \u0026amp; Ryan, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Milosavljevic et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kong et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Gziut \u0026amp; Thanacoody, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Together, these drugs illustrate the diversity of therapeutic strategies employed to counteract cerebral hyperexcitability.\u003c/p\u003e\u003cp\u003eTherefore, considering that local anesthetics administered in high doses or by continuous infusion can trigger seizure episodes, this study aimed to develop a levobupivacaine-induced seizure model and to analyze its behavioral and electrophysiological patterns, as well as the response to pharmacological control.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eA total of 72 adult male Wistar rats (200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g; 10 weeks old) were obtained from the Central Animal Facility of the Federal University of Par\u0026aacute;. The animals were housed in standard white cages (48 \u0026times; 38 \u0026times; 21 cm) under controlled environmental conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; 12/12 h light/dark cycle), with free access to food and water. All experimental procedures were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Animal Ethics Committee of the Federal University of Par\u0026aacute; (CEUA No. 6135040822). All necessary measures were taken to minimize animal suffering and distress.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Drugs\u003c/h2\u003e\u003cp\u003eKetamine was purchased from K\u0026ouml;nig and xylazine from Vall\u0026eacute;e, while levobupivacaine (LBE) was obtained from Crist\u0026aacute;lia Laboratory. Anticonvulsant compounds were sourced from different suppliers: phenobarbital (PHB) from Aventis Pharma, phenytoin (PHT) and diazepam (DZP) from Uni\u0026atilde;o Qu\u0026iacute;mica, and injectable sodium valproate (Depacon) from Abbott Laboratories do Brasil Ltda. (S\u0026atilde;o Paulo, Brazil).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experimental Design\u003c/h2\u003e\u003cp\u003eThe study comprised three experiments. In Experiment 1, seizure-related behavior was described in the LBE group (n\u0026thinsp;=\u0026thinsp;9), which received a single intraperitoneal (i.p.) dose of 20 mg/kg. Behavioral activity was assessed by measuring the latency to onset of convulsive behavior (in seconds).\u003c/p\u003e\u003cp\u003eIn Experiment 2, rats were randomly assigned to receive either vehicle (control) or levobupivacaine (20 mg/kg i.p.; n\u0026thinsp;=\u0026thinsp;9). Electrophysiological recordings were obtained from the CA1 region of the hippocampus to characterize the recorded traces.\u003c/p\u003e\u003cp\u003eIn Experiment 3, animals were treated with four anticonvulsant drugs (n\u0026thinsp;=\u0026thinsp;9 per group), following the protocol described by Hamoy et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e): (a) diazepam (DZP, 10 mg/kg i.v.), (b) phenobarbital (PHB, 10 mg/kg i.v.), (c) phenytoin (PHT, 10 mg/kg i.v.), and (d) sodium valproate (VPA, 10 mg/kg i.v.). In all groups, hippocampal recordings were performed. All drugs were administered via intravenous injection into the lateral tail vein. The vehicle group received 0.9% saline solution in a volume (ml) equivalent to body weight (kg).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Description of Seizure-Related Behavior\u003c/h2\u003e\u003cp\u003eSeizure-related behavior was observed following LBE administration. Seizure latency was recorded, and behavioral modifications were classified into four rapidly evolving, clinically identifiable stages: (a) bristling of whiskers, akinesia, and motionless staring; (b) generalized tremor without loss of the righting reflex; (c) generalized clonus with loss of the righting reflex; (d) clonic spasms with labored breathing, followed by respiratory arrest.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Hippocampal Recordings and Data Analysis\u003c/h2\u003e\u003cp\u003eHippocampal recordings were obtained using the procedures described by Quirino et al. (2024). Animals were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.) and xylazine hydrochloride (5 mg/kg, i.p.) and, after the abolition of the interdigital reflex, placed in a stereotaxic apparatus. The skull was exposed, and stainless-steel electrodes (0.03 mm in diameter, 3.4 mm in length), insulated with Teflon except for a 0.5 mm exposed tip, were implanted for signal acquisition. Electrodes were positioned in the CA1 region of the hippocampus according to the following stereotaxic coordinates: bregma \u0026minus;\u0026thinsp;3.00 mm, 1.6 mm lateral, and 3.40 mm dorsoventral. Five days after surgery, animals were treated with LBE, and electrodes were connected to a high-gain amplifier (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRecordings were performed following a standardized protocol. Animals were gently restrained for 10 minutes to allow adaptation and to minimize artifacts. Baseline hippocampal activity was recorded for 10 minutes and used as control in the analyses. Immediately after LBE administration, hippocampal activity was recorded for 10 minutes. Since LBE-induced seizures progressed rapidly to respiratory arrest, animals were euthanized at this stage to prevent further suffering.\u003c/p\u003e\u003cp\u003eFor evaluation of LBE-induced seizures controlled by anticonvulsants, animals were treated following a similar protocol. After receiving one of the anticonvulsant drugs (DZP, PHB, PHT, or VPA) via intravenous injection into the lateral tail vein, immediately after LBE administration (i.p.), hippocampal recordings were performed for 10 minutes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Data Analysis\u003c/h2\u003e\u003cp\u003eRecordings were obtained using a differential amplifier with high input impedance in AC mode (Grass Technologies, Model P511), adjusted with a 0.3 Hz\u0026ndash;3 kHz band-pass filter. Signals were monitored with an oscilloscope (Protek, Model 6510) and continuously digitized at a rate of 1 kHz by a computer equipped with a data acquisition board (National Instruments, Austin, TX).\u003c/p\u003e\u003cp\u003eSeizure characterization was performed using Python programming language (version 2.7) and the Signal\u0026reg; 3.0 software. Signal acquisition analyses were conducted in the frequency range up to 40 Hz, subdivided into theta (4\u0026ndash;8 Hz), beta (12\u0026ndash;28 Hz), and gamma (28\u0026ndash;40 Hz) bands, to interpret the dynamics of seizure development (Hamoy et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Euthanasia\u003c/h2\u003e\u003cp\u003eAfter completion of the experiments, animals were euthanized with a lethal intraperitoneal injection of ketamine (300 mg/kg) combined with xylazine hydrochloride (30 mg/kg, i.p.) and diazepam (10 mg/kg, i.p.). This procedure was necessary to prevent animal suffering.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e\u003cp\u003eResults were subjected to descriptive statistics, expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance (ANOVA) was performed, followed by Tukey\u0026rsquo;s post hoc test. Data were analyzed using GraphPad Prism, version 8 (GraphPad Software Inc.). Statistical significance was considered at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Seizure-Related Behavior Induced by LBE\u003c/h2\u003e\u003cp\u003eSeizure-related behavior was observed after intraperitoneal injection of LBE at a dose of 20 mg/kg. Animals exhibited continuous and progressive seizure stages. The mean latency for the first behavioral change\u0026mdash;whisker bristling, akinesia, and motionless posture\u0026mdash;was 119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51 s, followed by generalized tremor without loss of the righting reflex (stage 2) at 154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;12.29 s. Generalized clonic seizures with transient loss of the righting reflex (stage 3) occurred at 175.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.44 s, and tonic\u0026ndash;clonic seizures with labored breathing, followed by respiratory arrest (stage 4), were observed at 225.1\u0026thinsp;\u0026plusmn;\u0026thinsp;28.46 s (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\u003eMean latency for the onset of behaviors observed after levobupivacaine treatment in rats.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"left\" 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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBehavior\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBristling of whiskers, akinesia and motionless staring\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGeneralized tremor without loss of righting reflex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGeneralized clonus with transient loss of righting reflex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTonic-clonic seizure with labored breathing, followed by respiratory arrest\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLatency (s) (average and standard deviation)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51 (s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;12,29 (s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e175.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.44 (s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e225.1\u0026thinsp;\u0026plusmn;\u0026thinsp;28.46 (S)\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\u003eRecordings from the control group showed low-amplitude signals averaging 0.1 mV, with the highest energy concentrated below 10 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, the LBE group exhibited hippocampal alterations with cyclic peaks exceeding 0.4 mV, characteristic of burst potentials and indicative of seizure activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, left). The LBE group displayed a characteristic convulsive pattern, with distinct amplitude variations during ictal and interictal periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, center). During ictal periods, characteristic graphoelements of seizure syndromes (spike\u0026ndash;wave and polyspike complexes) were identified in the hippocampus. The spectrogram demonstrated seizure power and the rapid progression to respiratory arrest, which reduced spectrogram power (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, right).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring seizures, analysis of ictal and interictal periods induced by LBE injection demonstrated power fluctuations. The power spectral density (PSD) plot revealed higher power across all frequency bands during the ictal period compared with both the LBE baseline and the interictal recording. The largest power during the ictal state occurred in the theta (4\u0026ndash;8 Hz), beta (12\u0026ndash;28 Hz), and gamma (28\u0026ndash;40 Hz) ranges (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eSignificant between-group differences were observed across the frequency range up to 40 Hz. The control group showed a mean power of 0.259\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0275 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;, similar to the interictal period (p\u0026thinsp;=\u0026thinsp;0.972). The ictal period exhibited higher total spectral power (3.307\u0026thinsp;\u0026plusmn;\u0026thinsp;1.044 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) than all other conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFor theta oscillations, the control group (0.0802\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0109 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) did not differ from the interictal period (p\u0026thinsp;=\u0026thinsp;0.998) but was lower than the remaining groups. The ictal period averaged 1.344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.278 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;, exceeding all others (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor beta oscillations, the control group had a mean of 0.03797\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00578 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;, comparable to the interictal group (p\u0026thinsp;=\u0026thinsp;0.916) yet lower than the others. Mean power during the ictal period (1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.166 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was higher than in all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eFor gamma oscillations, the control group (0.0080\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0014 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was similar to the interictal group (p\u0026thinsp;=\u0026thinsp;0.983) but lower than the remaining groups. Mean power during the ictal period (0.228\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) exceeded all other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eAnticonvulsants were tested to determine whether they could reduce or abolish alterations in linear power recorded in the hippocampus by analyzing oscillatory power up to 40 Hz and in the beta range (12\u0026ndash;28 Hz). For this purpose, following intraperitoneal administration of levobupivacaine, PHT, PHB, DZP, and VPA were administered. Hippocampal recordings showed that the lowest amplitude was observed in the DZP group, while DZP and PHB provided the most effective seizure control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e\u003cp\u003eTo evaluate anticonvulsant activity, mean linear power was compared across groups. The control group showed 0.259\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0275 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;, which was similar to the LBE\u0026thinsp;+\u0026thinsp;PHB group (p\u0026thinsp;=\u0026thinsp;0.996) and the LBE\u0026thinsp;+\u0026thinsp;DZP group (p\u0026thinsp;=\u0026thinsp;0.999). The group treated with LBE alone (2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.751 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) exhibited higher values than all other groups. The LBE\u0026thinsp;+\u0026thinsp;PHT group (1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.461 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was similar to the LBE\u0026thinsp;+\u0026thinsp;VPA group (p\u0026thinsp;=\u0026thinsp;0.963). The LBE\u0026thinsp;+\u0026thinsp;PHB group (0.345\u0026thinsp;\u0026plusmn;\u0026thinsp;0.081 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was comparable to the LBE\u0026thinsp;+\u0026thinsp;DZP group (p\u0026thinsp;=\u0026thinsp;0.999) and to the LBE\u0026thinsp;+\u0026thinsp;VPA group (p\u0026thinsp;=\u0026thinsp;0.0508) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eTo further assess anticonvulsant activity, mean linear power in the beta range was compared across groups. The control group (0.0379\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00578 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was similar to the LBE\u0026thinsp;+\u0026thinsp;PHB group (p\u0026thinsp;=\u0026thinsp;0.989) and the LBE\u0026thinsp;+\u0026thinsp;DZP group (p\u0026thinsp;=\u0026thinsp;0.999). The group treated with LBE alone (0.718\u0026thinsp;\u0026plusmn;\u0026thinsp;0.114 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) exhibited higher values than all other groups. The LBE\u0026thinsp;+\u0026thinsp;PHT group (0.061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0117 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was like the LBE\u0026thinsp;+\u0026thinsp;VPA group (p\u0026thinsp;=\u0026thinsp;0.996) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe levobupivacaine group exhibited a convulsive state with amplitude oscillations, alternating between ictal and interictal periods, similar to the experimental model of lidocaine-induced seizures in rats standardized by Santos et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). That study demonstrated a behavioral and electroencephalographic progression that also differentiated ictal from interictal phases. In this context, lidocaine at toxic levels (60 mg/kg, i.p.) induced seizures defined by behavioral stages ranging from initial akinesia to generalized tonic\u0026ndash;clonic seizures. In the EEG, epileptiform alterations were observed, including high-amplitude, high-frequency discharges during the ictal phase, interspersed with periods of interictal activity. A comparable pattern was observed in the LBE group, where ictal activity mirrored that described in human seizure conditions.\u003c/p\u003e\u003cp\u003eSimilarly, Azevedo et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported in a rat model of caffeine-induced seizures that beta-band activity (12\u0026ndash;28 Hz) increased with high-frequency oscillations during the ictal state, corroborating our findings. In our study, ictal and interictal phases were clearly distinguished, with repeated sharp waves and high-amplitude discharges, supporting the concept that both LBE and caffeine generate electrodynamic seizure patterns with comparable detectability. This validates their use as models for studying electrophysiological seizure markers. Such evidence reinforces that convulsant mechanisms, whether via adenosine antagonism with caffeine or sodium channel blockade with LBE, can be reliably detected in animal models.\u003c/p\u003e\u003cp\u003eThis observation is in line with Hamoy et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), in which intraperitoneal infusion of cunaniol elicited interictal activity characterized by interspersed spikes preceding ictal episodes, correlating directly with behavioral patterns. Comparatively, in the acute seizure model induced by pentylenetetrazol (PTZ), (de Ara\u0026uacute;jo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that gamma oscillation power undergoes significant alterations during PTZ-induced seizures, with a reduction in gamma power noted during the postictal period. In contrast, our study revealed that gamma-band power increased markedly during ictal episodes, indicating intense neural activity typical of seizure onset. During interictal and control phases, gamma power remained low and stable, clearly distinguishing physiological states (control/interictal) from the pathological state (ictal) in terms of high-frequency activity. Supporting this, Zou et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) showed that oral administration of ciprofloxacin (CPC) for 14 days at therapeutic doses in rats increased susceptibility to seizures when PTZ was administered at higher doses. Interestingly, cortical CPC accumulation was not elevated, suggesting that seizure sensitization resulted from interference with GABA-A receptors and enhanced NMDA activity. Given the similarity between LBE- and PTZ-induced discharges, CPC sensitization may increase the risk of severe seizure episodes.\u003c/p\u003e\u003cp\u003eNeurotoxicity induced by LBE, particularly under continuous infusion or overdose, is characterized by multiple spike-and-slow-wave complexes, as demonstrated in our results. Based on this, Noji et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported a cumulative threshold dose of 28.63 mg/kg for LBE, which is considered relatively low compared with other agents. In contrast, bupivacaine displays a more specific toxicity profile. Lai (2022) reported activation of the NLRP3 inflammasome, increased caspase-1 activity, and GSDMD-N expression. Additionally, Yilmaz, Tepe, and Uludağ (2023) observed in rats that renal impairment alters pharmacokinetics, enhancing central nervous system toxicity due to prolonged exposure. This was associated with activation of TRPM2 (Transient Receptor Potential Melastatin 2) triggered by oxidative stress and mitochondrial dysfunction, along with increased Reelin expression leading to synaptic dysfunction and impaired neural plasticity. Collectively, these findings demonstrate that bupivacaine exhibits higher toxicity levels than LBE.\u003c/p\u003e\u003cp\u003eDuring the onset of behavioral alterations, a clear temporal progression of seizure stages was identified: 119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51 s \u0026ndash; Stage 1; 154.8\u0026thinsp;\u0026plusmn;\u0026thinsp;12.29 s \u0026ndash; Stage 2; 175.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.44 s \u0026ndash; Stage 3; and subsequently, tonic\u0026ndash;clonic seizures with labored breathing and respiratory arrest (Stage 4). This corroborates Noji et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), who reported similar transitions from Stages 1\u0026ndash;3 accompanied by the onset of ictal discharges (\u0026ge;\u0026thinsp;100 mV in the EEG at \u0026ge;\u0026thinsp;1 Hz). Similarly, Ohmura et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and Cheng et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) noted the predominance of Stages 2 and 3 (tremors and clonic seizures with loss of posture), as well as respiratory and circulatory collapse during Stage 4.\u003c/p\u003e\u003cp\u003eIn this context, attempts to increase the seizure threshold have included pharmacological interventions. Tanaka et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) demonstrated that dexmedetomidine prolonged the time and dose required for seizure onset, although progressive stages (prodromal signs, tremors, clonic, and tonic\u0026ndash;clonic seizures) were still observed, consistent with our findings. Likewise, Oda and Ikeda (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) used lipid emulsion with the same objective and successfully delayed seizure onset. More specifically, seizure initiation was postponed; however, progression still predominated from Stage 2 onward.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTherefore, the findings demonstrate that levobupivacaine, compared to other seizure models, consistently reproduces behavioral and electroencephalographic patterns characteristic of epileptic seizures, clearly distinguishing the ictal and interictal phases, with specific alterations in the fast bands. Furthermore, it was observed to have a relatively low toxic threshold, with neurotoxicity associated with multiple mechanisms, including mitochondrial dysfunction, inflammatory activation, and oxidative stress, making it more harmful than bupivacaine in certain contexts. It was also observed that anticonvulsants showed differentiated responses, with diazepam and phenobarbital demonstrating the greatest efficacy, significantly abolishing epileptiform discharges. In contrast, phenytoin and valproate showed only partial effect, attenuating but not completely reversing ictal activity, thus being less effective and, in some cases, refractory to immediate seizure control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: A.L. and M.H. Performed the experiments: A.L, D.B.A., L.E.Q., C.A.P., T.S.R. and M.H. Writing-original draft and editing: all authors. Financial support and administrative support: M.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Coordination for the Improvement of Higher Education Personnel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the Coordination for the Improvement of Higher Education Personnel (Brazilian CAPES), Amazon Foundation for Support of Studies and Research of the State of Par\u0026aacute; (FAPESPA), post-graduation in pharmacology and biochemistry of Federal University of Par\u0026aacute; (PPGFARMABIO). The authors also thank the students and staff of the Laboratory of Toxicology of Natural Products (UFPA \u0026ndash; Bel\u0026eacute;m) for developing the techniques that allowed the evaluation of electrophysiological activity.\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Accordance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were approved by the Animal Use Ethics Committee of the Federal University of Par\u0026aacute; (CEUA/UFPA, Protocol No. 6135040822). Experimental research on vertebrates was conducted in accordance with institutional, national, and international ethical guidelines for animal research and complies with the principles of the Basel Declaration (https://animalresearchtomorrow.org/en).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants were involved. The study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement; therefore, this item is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAS statement request\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available in the public repository at the following link: https://drive.google.com/file/d/1IzKv17hS0-QHNk97laZWnfFttXH_xXPk/view?usp=sharing.\u003cbr\u003eThe datasets support the findings of this study and include the original electrophysiological and behavioral data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\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\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbut, Y. C., Turkmen, A. Z., Midi, A., Eren, B., Yener, N., \u0026amp; Nurten, A. (2015). Efeitos neurot\u0026oacute;xicos de levobupivaca\u0026iacute;na e fentanil sobre a medula espinhal de ratos [Neurotoxic effects of levobupivacaine and fentanyl on rat spinal cord]. Revista brasileira de anestesiologia, 65(1), 27\u0026ndash;33.\u003c/li\u003e\n \u003cli\u003eAlthaus, A. L., Ackley, M. A., Belfort, G. M., Gee, S. M., Dai, J., Nguyen, D. P., Kazdoba, T. M., Modgil, A., Davies, P. A., Moss, S. J., Salituro, F. G., Hoffmann, E., Hammond, R. S., Robichaud, A. J., Quirk, M. C., \u0026amp; Doherty, J. J. (2020). 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Effect of lipid emulsion on the central nervous system and cardiac toxicity of bupivacaine and levobupivacaine in awake rats.\u0026nbsp;Journal of anesthesia,\u0026nbsp;27(4), 500\u0026ndash;504.\u003c/li\u003e\n \u003cli\u003eOhmura, S., Kawada, M., Ohta, T., Yamamoto, K., \u0026amp; Kobayashi, T. (2001). Systemic toxicity and resuscitation in bupivacaine-, levobupivacaine-, or ropivacaine-infused rats.\u0026nbsp;Anesthesia and analgesia,\u0026nbsp;93(3), 743\u0026ndash;748.\u003c/li\u003e\n \u003cli\u003ePaxinos, G., \u0026amp; Franklin, K. B. (2019). Paxinos and Franklin\u0026apos;s the mouse brain in stereotaxic coordinates. Academic press.\u003c/li\u003e\n \u003cli\u003ePostnikova, T. Y., Trofimova, A. M., Zakharova, M. V., Nosova, O. I., Brazhe, A. R., Korzhevskii, D. E., Semyanov, A. V., \u0026amp; Zaitsev, A. V. (2022). Delayed Impairment of Hippocampal Synaptic Plasticity after Pentylenetetrazole-Induced Seizures in Young Rats.\u0026nbsp;International Journal of Molecular Sciences,\u0026nbsp;23(21), 13461.\u003c/li\u003e\n \u003cli\u003eReddy, B. S., Gaude, Y. K., Vaidya, S., Kini, G. K., Budania, L. S., \u0026amp; Eeshwar, M. V. (2021). Effect of dexmedetomidine on characteristics of ultrasound-guided supraclavicular brachial plexus block with levobupivacaine-A prospective double-blind randomized controlled trial.\u0026nbsp;Journal of anaesthesiology, clinical pharmacology,\u0026nbsp;37(3), 371\u0026ndash;377.\u003c/li\u003e\n \u003cli\u003eRosenthal E. S. (2021). Seizures, Status Epilepticus, and Continuous EEG in the Intensive Care Unit. Continuum (Minneapolis, Minn.), 27(5), 1321\u0026ndash;1343.\u003c/li\u003e\n \u003cli\u003eSamanta D. (2021). Epilepsy in Angelman syndrome: A scoping review.\u0026nbsp;Brain \u0026amp; development,\u0026nbsp;43(1), 32\u0026ndash;44.\u003c/li\u003e\n \u003cli\u003eSantos, G. F. S., Ferreira, L. O., Gerrits Mattos, B., Fidelis, E. J., de Souza, A. S., Batista, P. S., Manoel, C. A. F., Cabral, D. A. C., J\u0026oacute;ia de Mello, V., Favacho Lopes, D. C., \u0026amp; Hamoy, M. (2020). Electrocorticographic description of the effects of anticonvulsant drugs used to treat lidocaine-induced seizures. Brain and behavior, 11(2), e01940.\u003c/li\u003e\n \u003cli\u003eShakerdi, L., \u0026amp; Ryan, A. (2023). Drug-induced hyperammonaemia.\u0026nbsp;Journal of clinical pathology,\u0026nbsp;76(8), 501\u0026ndash;509. https://doi.org/10.1136/jcp-2022-208644\u003c/li\u003e\n \u003cli\u003eSteverink, J. G., Piluso, S., Malda, J., \u0026amp; Verlaan, J. J. (2021). Comparison of in vitro and in vivo Toxicity of Bupivacaine in Musculoskeletal Applications. 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Clinical EEG and neuroscience, 55(2), 278\u0026ndash;282. https://doi.org/10.1177/15500594231183554\u003c/li\u003e\n \u003cli\u003eUzun, U., \u0026amp; İdin, K. (2025). Comparison of Onset Times and Hemodynamic Changes of Bupivacaine and Levobupivacaine Used in Spinal Anesthesia. Anatolian Journal of General Medical Research, 35(2), 116-121. https://doi.org/10.4274/anatoljmed.2025.75537\u003c/li\u003e\n \u003cli\u003eYang, Y., Wang, C., Liu, J., Liao, D., Zhang, W., \u0026amp; Zhou, C. (2022). QX-OH/Levobupivacaine: A Structurally Novel, Potent Local Anesthetic Produces Fast-Onset and Long-Lasting Regional Anesthesia in Rats. Journal of pain research, 15, 331\u0026ndash;340. .\u003c/li\u003e\n \u003cli\u003eYılmaz, N., Tepe, M., \u0026amp; Uludağ, \u0026Ouml;. (2023). Examination of the effect of bupivacaine on brain tissue in rats with induced experimental renal failure: Neurotoxicity of bupivacaine in renal failure.\u0026nbsp;Journal of Surgery and Medicine,\u0026nbsp;7(9), 598\u0026ndash;601.\u003c/li\u003e\n \u003cli\u003eYoshimoto, M., Horiguchi, T., Kimura, T., \u0026amp; Nishikawa, T. (2017). Recovery From Ropivacaine-Induced or Levobupivacaine-Induced Cardiac Arrest in Rats: Comparison of Lipid Emulsion Effects.\u0026nbsp;Anesthesia and analgesia,\u0026nbsp;125(5), 1496\u0026ndash;1502.\u003c/li\u003e\n \u003cli\u003eZhang, M., Kou, L., Qin, Y., Chen, J., Bai, D., Zhao, L., Lin, H., \u0026amp; Jiang, G. (2022). A bibliometric analysis of the recent advances in diazepam from 2012 to 2021.\u0026nbsp;Frontiers in pharmacology,\u0026nbsp;13, 1042594.\u003c/li\u003e\n \u003cli\u003eZhvania, M. G., Sharikadze, I., Japaridze, N., Tizabi, Y., Rzayev, F., Gasimov, E., \u0026amp; Lobzhanidze, G. (2025). Status epilepticus alters hippocampal ultrastructure in kainic acid rat model.\u0026nbsp;Tissue \u0026amp; cell,\u0026nbsp;94, 102789.\u003c/li\u003e\n \u003cli\u003eZou, S., Li, Y., Zou, Q., Yang, M., Li, H., Niu, R., Lai, H., Wang, J., Yang, X., \u0026amp; Zhou, L. (2024). Gut microbiota and serum metabolomic alterations in modulating the impact of fecal microbiota transplantation on ciprofloxacin-induced seizure susceptibility. Frontiers in microbiology, 15, 1403892. https://doi.org/10.3389/fmicb.2024.1403892\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Levobupivacaine, Seizure model, Status epilepticus, Electrophysiology, Anticonvulsants, Wistar rats","lastPublishedDoi":"10.21203/rs.3.rs-7973054/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7973054/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStatus epilepticus (SE) remains a life-threatening neurological emergency driven by prolonged, excessive neuronal discharges. At toxic concentrations, levobupivacaine (LBE), a potent local anesthetic, can provoke seizures and thus serves as a practical model to study seizure generation and therapeutic response. We established an LBE-induced seizure model in adult Wistar rats and combined behavioral scoring, hippocampal field recordings, and pharmacological intervention. Following intraperitoneal LBE (20 mg/kg), animals progressed reproducibly through four behavioral stages (mean latencies: 119.6 s, 154.8 s, 175.9 s, and 225.1 s), ending in tonic\u0026ndash;clonic events complicated by respiratory failure. Electrophysiological traces showed alternating ictal and interictal epochs with high amplitude burst discharges and significant increases in spectral power across theta, beta, and gamma bands. Intravenous administration of diazepam and phenobarbital markedly suppressed ictal discharges; phenytoin and valproate yielded only partial attenuation. Collectively, the LBE model reproduces key behavioral and electrographic hallmarks of convulsive SE and provides a reproducible platform for probing convulsant neurotoxicity and for preclinical screening of anticonvulsant strategies.\u003c/p\u003e","manuscriptTitle":"Levobupivacaine induces seizures in Wistar rats through behavioral, electrophysiological, and pharmacological modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 03:33:33","doi":"10.21203/rs.3.rs-7973054/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T15:24:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T05:48:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136524235494025332062696633358041515697","date":"2025-11-22T14:56:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42500789626419495147544816515471080069","date":"2025-11-19T04:26:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T05:54:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37348099762239021288433610585284677298","date":"2025-11-05T19:46:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T18:43:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-05T18:40:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-01T10:39:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-31T19:01:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Animals","date":"2025-10-31T18:57:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-animals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Animals](https://link.springer.com/journal/44338)","snPcode":"44338","submissionUrl":"https://submission.springernature.com/new-submission/44338/3","title":"Discover Animals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d13fd820-ae03-4da8-8648-9c1caf1502b3","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T12:57:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 03:33:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7973054","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7973054","identity":"rs-7973054","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}