Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study

Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study | 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 Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study Chao Gong, Xunzhong Qi, Luchuan Wang, Pei Zeng, Beibei Lian, Jiahao Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6659867/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 Objective Ischemia and hypoxia are frequently associated with epileptic characteristics, and irregular calcium metabolism can exacerbate neuronal damage. To investigate calcium homeostasis and its effect on neuronal injury, hippocampal HT22 cells were subjected to oxygen-glucose deprivation (OGD), followed by reoxygenation and culturing in Mg 2+ -free medium, to mimic cerebral ischemic and hypoxic brain injury combined with epilepsy. Methods An oxygen-glucose deprivation combined with epileptiform discharge (OGD&ED) model was established to simulate the pathological state of cerebral ischemia-hypoxia combined with epilepsy. The model was evaluated based on cell viability and Ca²⁺ overload, and the optimal induction conditions were determined as 3-hour OGD, 3-hour culture without Mg²⁺, and 8-hour reoxygenation. A calcium-sensing receptor (CaSR) agonist (R568) and inhibitor (NPS-2143), combined with a Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) inhibitor (KN-93), were used to study the regulatory effect of Ca²⁺ overload on the mitochondrial apoptotic pathway. The apoptosis rate, mitochondrial damage, intracellular Ca²⁺ concentration, and changes in the expression of calcium-related proteins were detected. Results Compared with the single OGD group or the ED group, the OGD&ED group showed a significant increase in Ca²⁺ influx, more severe cell and mitochondrial damage, and a higher apoptosis rate. The CaSR agonist R568 aggravated Ca²⁺ overload, while the inhibitor NPS-2143 and the CaMKII inhibitor KN-93 effectively inhibited Ca²⁺ influx. Ca²⁺ overload activated the mitochondrial apoptotic pathway, resulting in abnormal expression of apoptosis-related proteins and further aggravating neuronal injury. Conclusion In the OGD&ED model with an intervention time of OGD3h, Mg 2+ -free, and reoxygenation8h, Ca 2+ overload exacerbates the damage to cells and mitochondria and promotes apoptosis through the mitochondrial pathway. Calcium overload Oxygen-glucose deprivation Epileptiform discharge Mitochondria CaSR CaMKⅡ Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cerebral palsy (CP) is a group of lifelong central motor disorder syndromes that significantly affect early childhood survival and quality of life [ ] . According to population-based studies, the global prevalence of CP ranges from 0.1–0.4%, making it one of the most common conditions threatening early childhood survival [ ] . Epilepsy is a common clinical manifestation in children with CP and is widely regarded as an indicator of CP severity. While the prevalence of epilepsy in the general population is estimated to be around 0.4–0.8%, it is notably higher among children with CP, with research from our group suggesting a prevalence ranging from 31.6–46.5% [ ] . This co-occurrence may be attributed to shared pathophysiological mechanisms associated with common etiologies and risk factors, resulting in the simultaneous onset of both CP and epilepsy [ , ] . Post-ischemic stroke epilepsy (PISE) refers to non-provoked epileptic seizures that occur two or more times (with an interval of 24 hours) in patients with no previous history of epilepsy, and these seizures are secondary to ischemic brain tissue damage caused by severe stenosis or occlusion of blood vessels [ ] . Ischemic stroke is a common cause of secondary epilepsy in the elderly aged over 60 worldwide, with an incidence rate as high as 25%-50%. Some studies have shown a long-term cumulative risk of PISE, with the proportion of late-onset epileptic seizures in all PISE patients being 2%-15% [ ] . Despite its prevalence, the exact mechanisms linking epilepsy to CP and stroke remain unclear. In many cases, the cause of epilepsy in children with CP and stroke is unknown. A key factor may be ischemia-reperfusion injury, which occurs after cerebral ischemia and glucose deprivation. In this scenario, the restoration of blood flow does not lead to recovery but instead results in greater dysfunction. The oxygen-glucose deprivation/reoxygenation (OGD/R) model effectively simulates this process. The epileptic discharge (ED) model, induced by incubation in Mg 2+ -free extracellular fluid, is a widely used in vitro model to replicate epileptic seizures. Studies have demonstrated that the absence of Mg 2+ leads to intracellular Ca 2+ influx, causing mitochondrial damage and ultimately resulting in cell death. Calcium homeostasis, serving as a key second messenger, plays a crucial role in maintaining neuronal function. An increase in intracellular Ca 2+ (often referred to as Ca 2+ overload) can trigger several neurotoxic mechanisms. Both cerebral ischemia-reperfusion injury and epilepsy can lead to Ca 2+ overload, which induces neuronal damage via mitochondrial dysfunction, activation of calcium-dependent degrading enzymes, and increased production of oxygen free radicals [ , ] . Mitochondria play essential roles in ATP synthesis, reactive oxygen species (ROS) production, Ca 2+ buffering, and apoptosis signaling. In neurons, mitochondrial Ca 2+ buffering is critical for maintaining neuronal excitability, synaptic transmission, and overall cell function [ ] . Mitochondrial Ca 2+ overload is a common pathological mechanism underlying neurological conditions, including mitochondria-related epilepsy and post-stroke epilepsy. Epilepsy can be caused by various neurological conditions, including stroke (11.4% [ ] ), cerebral palsy (31.6%-46.5% [3] ), and traumatic brain injury. Brain injury often leads to structural changes in neural networks and increased neuronal excitability, which heightens the risk of epilepsy [ , ] . Ischemia causes a deficiency in oxygen and glucose supply to the brain, and the longer the restoration of blood flow is delayed, the more severe the resulting dysfunction. This process is referred to as ischemia-reperfusion injury, which forms the basis for the development of the OGD/R model. The OGD/R model is widely used in research to simulate ischemic stroke in vitro, while the ED model, created by culturing cells in a Mg 2+ -free extracellular environment, is commonly used to simulate epileptic seizures [ ] . Studies have shown that a lack of intracellular Mg 2+ can lead to Ca 2+ influx, causing mitochondrial damage and subsequent cell death [ ] . Given the strong association between epilepsy and both CP and stroke, particularly in cases involving ischemic brain injury, there is a need for further research in this area. While the OGD/R model is commonly used in studies to investigate stroke, its application in simulating CP-related ischemic brain injury remains limited. This study aims to build on existing research by introducing a Mg 2+ -free intervention during the reoxygenation phase of OGD, thereby establishing an OGD&ED model that can serve as a reference for future investigations. Furthermore, both OGD/R and ED have been shown to cause intracellular Ca 2+ overload, leading to mitochondrial dysfunction and apoptosis. This study will explore the mechanisms underlying Ca 2+ overload in this OGD&ED model and provide insights into the potential therapeutic targets for mitigating neuronal damage. Epilepsy is one of the most common neurological disorders, affecting about 0.5%~1% of the world's population [ ] . Epilepsy is caused by the abnormal firing of neurons due to an imbalance of Ca 2+ in the neurons. Ca 2+ homeostasis, which includes Ca 2+ influx, efflux, caching, storage, and diffusion, plays a significant role in maintaining cell function and acts as a ubiquitous second messenger. The disruption of calcium homeostasis is a major initiator and activator of the cell death pathway [ , ] . Ca 2+ overload refers to the abnormal increase of intracellular Ca 2+ caused by various reasons, leading to abnormal phenomena of cell structure and function damage. Both cerebral ischemia-reperfusion and epilepsy can cause Ca 2+ overload, causing damage to nerve cells. Research has shown that it may involve three major mechanisms: mitochondrial dysfunction, activation of calcium-dependent degrading enzymes, and promoting oxygen-free radical generation [ ] . The calcium-sensing receptor (CaSR) plays a crucial role in calcium homeostasis. Abnormal CaSR function may lead to nervous system diseases, including epilepsy [ ] . Current research indicates that abnormal changes in the calmodulin-dependent protein kinase II (CaMKII) pathway, which is associated with epileptogenesis, lead to decreased CaMKII levels in multiple epileptic models [ ] . In this study, we first established a model to determine the optimal time for generating characteristic features of both OGD and ED conditions, then applied Ca 2+ regulators, including CaSR and CaMKII agonists and inhibitors, to investigate the effects of intracellular Ca 2+ overload on mitochondrial dysfunction and cell apoptosis, with a focus on the pro-apoptotic effects of the mitochondrial pathway. 2. Method and materials The whole experiment was shown in the flow chart, Fig. 1 . 2.1 Cell culture Mouse hippocampal neuron HT22 cells (RRID: CVCL_0321) were obtained from Shanghai Heisou Biotechnology, Shanghai, China. HT22 has not been listed as a commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC). Its passage number is limited to 5 times. The cell culture medium was prepared with 10% Fetal Bovine Serum (FBS, RRID: SCR_014489, Gibco, the United States) and 1% penicillin (cat. no. C0222, Beyotime, China). The Mg 2+ -free solution was prepared with NaCl (124.0 mmol/L), NaHCO3 (25.0 mmol/L), KCl (3.5mmol/L), CaCl2 (2.5 mmol/L), NaH2PO4 (1.2 mmol/L) and glucose (10.0 mmol/L), and the pH was adjusted to 7.4 with 1.0 mol/L HCl. 2.2 Model preparation To explore cell viability and Ca 2+ concentration in OGD&ED model cells, HT22 cells were subjected to OGD for 1 to 6 hours, followed by Mg 2+ -free incubation for 0 to 6 hours, and reoxygenation for 8, 12, and 24 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (cat. no. C0038, Beyotime, China), during both OGD and reoxygenation phases. Furthermore, the Fluo-4 AM probe (cat. no. S1060, Beyotime, China) was utilized to determine the intracellular Ca 2+ concentration over a 0 to 6-hour Mg 2+ -free. Ca 2+ overload in the subsequent studies were investigated under conditions that result in the highest Ca 2+ concentration. There were seven groups in the experiment, they were, 1. Control group: HT22 cells were conventionally cultured with normal oxygen and Mg 2+ with no treatment. Briefly, when the monolayer of HT22 cells in the petri dish reaches about 80% confluence, proceed with subculturing. Discard the supernatant, wash the cells with PBS 2–3 times, and add 2 mL of 0.25% trypsin solution for digestion. After 2 minutes, 1 mL of complete medium was added to stop the digestion, gently blew the adherent cells off to form a cell suspension, and transferred it to a 15 mL centrifuge tube, centrifuged at 1400 rpm for 5 minutes. The supernatant was discarded, complete medium was added to gently resuspend the centrifuged cells into a single-cell suspension, and then subcultured at a ratio of 1:3 into new culture dishes for continuous cultivation. 2. OGD group: HT22 cells in the logarithmic growth phase were quantified and seeded them into plates well and cultured in a regular medium until confluency, then the medium was switched to a sugar-free Dulbecco's Modified Eagle Medium (DMEM, cat. no. 11966025 Gibco, the United States), placed in an anaerobic tank, and incubated at 37℃ for hypoxia. After rinsing with sterile PBS, the medium was switched to a normal culture medium for reoxygenation with sugar-containing medium. 3. ED group: HT22 cells were incubated in a Mg 2+ -free extracellular solution until confluency, then the medium was switched to sugar-containing medium. 4. OGD&ED group: Upon reaching confluency, HT22 cells were cultured in sugar-free DMEM, placed in an anaerobic tank, incubated at 37℃ for hypoxia, followed by reoxygenation first with Mg 2+ -free extracellular solution, then with sugar-containing medium. 5. OGD&ED + R568 group: Similar to the OGD&ED group, but with 10 µmol/L CaSR agonist [R568 (cat. no. HY-10167, MCE, the United States)] added during the reoxygenation phase. 6. OGD&ED + NPS-2143 group: Based on the OGD&ED protocol, but with 10 µmol/L CaSR inhibitor [NPS-2143 (cat. no. HY-10007, MCE, the United States)] added during reoxygenation. 7. OGD&ED + KN-93 group: Following the OGD&ED procedure, with the addition of 10µmol/L CaMK II inhibitor [KN-93(cat. no. HY-15465, MCE, the United States)] during reoxygenation. 2.3 Cell activity Cell viability and proliferation were assessed using the CCK-8 assay as per manufacturer's instructions. Cells were seeded in 96-well plates at 5×10^4 cells/well. Post-treatment, 10 µL of CCK-8 solution was added to each well and incubated at 37℃ for 2 hours. Absorbance was measured at 450 nm using an enzymoleter, and cell viability was calculated and plotted. Intracellular Ca 2+ concentration measurement Cells were seeded onto 24-well plates and, upon reaching approximately 60% adhesion, they were rinsed with sterile PBS following the experimental protocol. Then, 250 µL of Fluo-4 dye solution was added to each well and incubated for 30 minutes at 37ºC in the dark. Post-incubation, the staining was examined under a fluorescence microscope. 2.4 The morphology of hippocampal neurons After the intervention was performed on approximately 50% of the adherent cells, the morphological changes were observed using an inverted phase contrast microscope. Cell images were taken using a camera under a microscope for further analysis. 2.5 Detection of intracellular ROS Cells were seeded onto 24-well culture plates. Following the intervention, the culture medium was removed, and fresh serum-free medium with 10µmoL/DCFH-DA probe (cat. no. S0034S, Beyotime, China) was added, then incubated at 37℃ for 20 minutes. The cells were washed three times with serum-free medium before being observed under a fluorescence microscope. 2.6 Morphology of mitochondria Cells were cultured in a dish and treated at 80% confluence. HT22 cells were trypsinized, washed with PBS, and centrifuged at 800 rpm for 10 min. The supernatant was discarded, and the cell pellet fixed in 2.5% glutaraldehyde for 24 hours, followed by 1% osmium tetroxide for 1 hour, washed once with PBS, dehydrated in an ethanol series, and embedded in EPON resin. Thin sections were stained with 1% uranyl acetate and lead citrate, and the mitochondrial structure observed by transmission electron microscopy. 2.7 Apoptosis assessment After the intervention, cells from the seven groups were collected, the culture medium was removed, and cells were washed with PBS, fixed with immunofixation solution for 30 minutes at room temperature, washed again with PBS, and treated with 0.3% Triton X-100 in PBS for 5 minutes at room temperature, followed by 2–3 PBS washes. The TUNEL (cat. no. MA0223, MeilunBio, China) reaction mixture was prepared as per the instructions, and 50 µL was added to each sample, covered with anti-evaporation film, and incubated in a humidified chamber at a constant temperature for 60 minutes in the dark. After incubation, cells were washed three times with PBS, stained with 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, cat. no. MA0128, MeilunBio, China) for 5 minutes in the dark, followed by three PBS washes. Images were collected using a fluorescence microscope. 2.8 Western blot assay In this study, we presented the full membranes with all target proteins and GAPDH together. Proteins were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked in TBST containing 5% skim milk powder at room temperature for 2 hours, followed by incubation with primary antibodies at 4°C: CaSR (1:2000, cat. no. AB_10646434, Proteintech, the United States), CaMKII (1:1000, cat. no. ab134041, Abcam, the United Kingdom), Bax (1:2000, cat. no. AB_2819658, Proteintech, the United States), Bcl2 (1:1000, cat. no. AB_2918886, Proteintech, the United States), Caspase 3 (1:1000, cat. no. 82202-1-RR, Proteintech, the United States), and GAPDH (1:50000, cat. no. 60004-1-Ig, Proteintech, the United States). After primary antibody incubation, the membrane was incubated with HRP-conjugated secondary antibodies (1:20000, cat. no. BA1070, Boster, China) at room temperature for 2 hours. Chemiluminescence signals were visualized using a chemiluminescence system, and the intensity of the bands was quantified using Image J software. 2.9 Immunofluorescence staining CaSR (1:150), CaMKII (1:250), Bcl2 (1:400), and Caspase 3 (1:50) in HT22 cells were detected by immunofluorescence labeling. Each group was fixed, permeabilized, blocked, and incubated with the first antibody at 4 ℃ overnight, followed by incubation with the fluorescent goat anti-rabbit secondary antibody (cat. no. BA1091 and BA1090, Boster, China) at 37 ℃ for 1.5 hours. 2.10 qRT-PCR analysis Cell samples were processed for qRT-PCR analysis using a cDNA kit (cat. no. RR036A and RR820A-200Rx, Takara, Japan) according to the provided guidelines. The qRT-PCR was performed on an automatic medical PCR instrument (SLAN-96P) using SYBR Green qPCR Master Mix. The reverse transcription step involved incubation at 95°C for 10 minutes, followed by an hour at 42°C. For the qPCR amplification, a Real-Time reaction mixture of 5µl was prepared in a total volume of 50µl, containing custom gene-specific primers for the target sequences. The qPCR amplification consisted of 40 cycles, with denaturation at 95°C for 15 seconds, followed by annealing at 56°C for 60 seconds. Primers for CaSR were 5′-ACCTTTACCTGTCCCCTGAA-3′ (Forward) and 5′-GGGCAACAAAACTCAAGGTG-3′ (Reverse); for CaMKII, 5'-GCGACCTGAAGCCTGAGAATCTG − 3'(Forward) and 5'-GTGTCCCTGCGAACCCAAACC-3' (Reverse). 2.11 Statistics Statistical analysis and plotting were performed using GraphPadPrism8.0.2 and SPSS18.0. Each experimental group for every detection index was repeated 3 to 5 times, and results were presented as mean ± standard deviation. Data were assessed for normality, using the Shapiro–Wilk test. All data showed a p > 0.05 for this, indicating they were normally distributed. The generalised ESD test was conducted to determine whether significant outliers existed. The test showed no outliers, and no data points were excluded from the analysis. Univariate analysis of variance and repeated measures analysis of variance were utilized for statistical analysis, with P < 0.05 was considered statistically significant. The data that support the findings of this study are openly available in Mendeley at https://data.mendeley.com/datasets/5699hyhccj/1 . 3 Results and discussion 3.1 Exploration of the OGD&ED Model Extensive prior research has been dedicated to identifying optimal durations for OGD in nerve cell OGD/Reoxygenation (OGD/R) models [ − ] . A comprehensive review of the current literature revealed that the most commonly employed OGD/R durations are 2 and 4 hours, with Mg 2+ -free conditions frequently applied for 3 hours [ ] . This insight underscores the importance of precisely calibrating intervention times to navigate the considerable variability observed in cellular responses across differing experimental setups and conditions. The CCK-8 assay relies on the oxidation of water-soluble tetrazolium salt-8, which is reduced by cellular dehydrogenases to form a soluble formazan dye to produce yellow coloration. This allows the measurement of cell metabolic activity by dehydrogenases in active mitochondria and allows a direct metric of cell viability. This assay is directly proportional to the count of viable cells. In this investigation, 18 specific time points were delineated across varying conditions (OGD periods from 1 to 6 hours, Mg 2+ -free periods from 0 to 6 hours, and reoxygenation intervals at 8, 12, and 24 hours) as illustrated in Fig. 2 A, to systematically evaluate cell viability using the CCK-8 assay. Initial analyses focused on discerning the impacts of various intervention intervals within the first hour, notably identifying a significant suppression of cell viability during the initial hour of Mg 2+ -free intervention. This observation hints at the potential of short-term Mg 2+ -free conditions during reoxygenation to induce epileptiform discharges in nerve cells, consequent to post-reperfusion injury, and its pronounced effects on cell viability. Moreover, the analysis revealed that reoxygenation curves for durations of 8, 12, and 24 hours exhibit convergence when OGD interventions are extended to 5 and 6 hours. This pattern suggests that the extended durations of OGD exert a pronounced inhibitory effect on cellular functions, thereby diminishing the relative influence of reoxygenation duration on cell viability. To optimally configure the OGD&ED model, it is crucial to pinpoint conditions that minimize cell viability while still allowing for significant recovery. This involves subjecting cells to OGD conditions devoid of glucose and oxygen over six distinct intervals. Following each interval, the culture medium was replaced with a standard solution lacking Mg 2+ for 0-6h, succeeded by reoxygenation phases lasting 8, 12, and 24 hours, respectively. Our experimental model also incorporated a control group comprising cells cultured in standard medium replete with glucose and oxygen. The influence of varying OGD durations, Mg 2+ -free conditions, and reoxygenation times on cell viability is systematically presented in Fig. 2 B, with a notable reduction in cell viability observed particularly during the shortest reoxygenation period of 8 hours compared to the longer spans of 12 and 24 hours. Statistical analyses confirmed significant disparities across different Mg 2+ -free intervention intervals and between groups subjected to varied durations of Mg 2+ deprivation. Collectively, the data from Fig. 2 A pinpoint the specific conditions of 3 hours of OGD followed by 8 hours of reoxygenation, in conjunction with 3 hours in a Mg 2+ -free medium, as markedly reducing cell viability. This finding, supported by additional data, underscores a significant decrease in viability immediately following Mg 2+ removal, with a continued decline observed across all subsequent periods. The delineated trend, with OGD duration as a variable, suggests an initial rise followed by a decline in cell viability, with the most significant effects observed at 3 hours of OGD and the most considerable impact of reoxygenation time at 8 hours, see Fig. 2 B. Accordingly, this 3-hour OGD period followed by 8 hours of reoxygenation has been chosen for further investigation into the ramifications of Mg 2+ -free conditions on intracellular Ca 2+ dynamics. 3.2 Intracellular Ca 2+ concentration and related protein expression in OGD&ED model Upon interruption of cerebral blood flow, mitochondria transition from anaerobic to aerobic metabolism, resulting in an increase in lactic acid production that leads to metabolic acidosis [ ] . This shift, coupled with a diminished energy reserve, prompts depolarization of the membrane potential and impairs the functionality of both the sodium-potassium pump (Na+-K+-ATPase) and the calcium pump (Ca 2+ -ATPase) at the cell surface [ − ] . Consequently, Na+/H + exchangers eject H + and admit an influx of Na+, facilitating the entry of Ca 2+ through the plasma membrane via Na+/Ca 2+ exchangers. Moreover, the inactivation of ATPase contributes to a decrease in Ca 2+ efflux and a restriction in Ca 2+ uptake by the endoplasmic reticulum, culminating in Ca 2+ overload within the cells and impacting the function of mitochondria [ ] . Ca 2+ is recognized as a pivotal factor in the initiation and progression of epilepsy, given its role in Ca 2+ -dependent signaling pathways that modulate neuronal activity in various ways, ultimately leading to seizures through the enhancement of either focal or generalized neuronal hyperexcitability [ , ] . Emerging evidence underscores the critical function of Ca 2+ signaling in neurons in relation to both seizures and epilepsy. The orchestration of extraneuronal seizure synchronization involves numerous Ca 2+ signaling pathways, and the interplay between mitochondrial Ca 2+ signaling and ROS may precipitate neuronal cell death and epileptic discharges [ ] . Employing a Ca 2+ fluorescence probe, intracellular Ca 2+ concentrations were quantified from 0 to 6 hours without Mg 2+ during a regimen of 3 hours of OGD followed by 8 hours of reoxygenation, juxtaposing these measurements against a control group. Findings (Fig. 2 C and D) revealed a notable increase in intracellular Ca 2+ at 3 hours culturing medium without Mg 2+ , with further elevation at 4 hours. These observations underscore the pronounced impact of OGD and subsequent reoxygenation, particularly during the Mg 2+ -depleted phase, on mitochondrial function and apoptosis, thereby significantly affecting cellular integrity. The apex of Ca 2+ levels at 3 hours post-OGD without Mg 2+ highlights the intervention's efficacy in instigating apoptosis via mitochondrial Ca 2+ overload. Further analysis demonstrated that Ca 2+ concentrations in the OGD&ED model significantly exceeded those in the control group, as well as in the groups subjected only to ED and OGD. This suggests that the OGD&ED model precipitates a greater influx of Ca 2+ , prompting a deeper examination of the relationship between Ca 2+ overload and apoptosis. This correlation emphasizes the necessity of understanding mitochondrial Ca 2+ dynamics as a potential mechanism underlying cell death and epileptiform activity in the context of cerebral ischemia and reperfusion. Fu et al. [ ] found that the frequency of the CC genotype in PISE patients was significantly higher than that in the non - epileptic control group, and the distribution of the rs2274924C allele was wider in PSE patients. The C allele of rs2274924 is associated with a lower level of magnesium ions in the serum, which increases the intracellular sodium ions, promotes sodium/calcium exchange, increases the concentration of intracellular calcium ions, enhances neuronal excitability, and induces epileptic seizures. It is thus inferred that the polymorphism of TRPM6 rs2274924 and the lower level of serum magnesium ions are potential predictors of post - stroke epilepsy. The expression of CaSR was higher than that in the ED and OGD groups, and the Ca 2+ concentration further increased or decreased after the application of CaSR agonists and inhibitors. CaSR is highly expressed in the nervous system and is involved in maintaining intracellular Ca 2+ homeostasis [ , ] . It is possible that CaSR mediates more Ca 2+ influx. Ca 2+ is a ligand of CaSR, which can act as the first messenger to activate the expression of CaSR in nerve cells, and then induce Ca 2+ overload, resulting in neuronal apoptosis after OGD [ ] . As a key factor in the regulation of Ca 2+ signaling to maintain intracellular Ca 2+ homeostasis, CaMKⅡ can be activated when the intracellular Ca 2+ concentration is induced by neuronal activity [ , ] , and then induce neuronal Ca 2+ overload and induce neuronal apoptosis. However, our study (Fig. 3 B) showed that the expression of CaMKⅡ protein and mRNA was higher than that of ED group, but lower than that of the OGD group. In theory, increased CaMKII activity may underlie seizures, but multiple studies have shown a direct link between reduced CaMKII activity and epilepsy. It has been shown that reduced CaMKII activity and expression represents a homeostasis mechanism that reduces the effects of Ca 2+ overload caused by excess neuronal activity during seizures [ ] . Therefore, we speculated that the occurrence of ED in the OGD&ED model led to the decrease of CaMKⅡ expression after OGD modeling, but there is no doubt that this is in conflict with the traditional theory that the increase of CaMKII activity may be the basis of epileptic seizures. However, the increase of CaMKII after OGD/R is unquestioned, and CaMKII activation is considered to be a key factor in ischemia-induced neuronal cell death. In ischemic stroke, there is compelling evidence to support rapid activation of CAMKII immediately after hypoxia/reperfusion injury. Activated CaMKII plays a key role in mediating excitotoxic-induced neuronal death [ ] . Cerebral ischemia-induced translocation of CaMKII into the synaptic membrane may increase neuronal firing rate and Ca 2+ inflow. In addition, current studies have shown that the inactivation of CaMKⅡ can inhibit calcium overload and thus play a protective role in cells. In addition, CaSR and CaMKⅡ interact with each other [ ] . Studies have found that CaSR activation can up-regulate the expression activity of CaMKⅡ, activate the NLRP3 inflammasome, and aggravate brain injury, while the application of NPS-2143 and KN-93 can inhibit the expression of CaSR and CaMKⅡ, and alleviate OGD injury [ , ] . 3.3 Cell status and mitochondria damage In the normal group, cells displayed smooth, full surfaces and matured into tight neural networks. In contrast, cells in both the ED and OGD groups exhibited disintegration, collapse, with thin protrusions, atrophy, fractures, necrosis, and reduced adhesion, leading to detachment. This degradation was more pronounced in the OGD&ED group, where cell shedding increased. The use of R568 during reoxygenation, which increased intracellular Ca 2+ and protein expression, aggravated the damage. However, treatments with NPS-2143 and KN-93 improved cell morphology, adhesion, and growth (Fig. 4 A). Calcium acts as a critical second messenger in various cellular processes, including neuronal growth, differentiation, and synaptic plasticity. However, when calcium homeostasis is disrupted (for instance, during pathological states leading to calcium overload), it can inhibit neurite outgrowth, alter axonal transport, and impair synaptic formation, ultimately affecting neuronal cell survival and growth. Ureshino’s study notes that disruptions in Ca 2+ signaling, often due to overload, can lead to enhanced vulnerability to degeneration by activating destructive enzymes and impairing cellular functions. This disruption is particularly detrimental in neurons, where precise Ca 2+ regulation is crucial for normal function and survival [ , ] . Mitochondrial observations echoed these findings; normal mitochondria were noted in the control group, whereas the ED and OGD groups showed mitochondrial deformation, irregular shapes, and cristae damage. The OGD&ED group faced severe mitochondrial damage, including mitochondriolysis and balloon-like transformations. R568 treatment intensified these effects by raising intracellular Ca 2+ levels, while NPS-2143 and KN-93 treatments alleviated the damage (Fig. 4 C). Similarly, ROS levels in the OGD&ED group were significantly higher than in the control, ED, and OGD groups, with R568 further elevating ROS levels. Conversely, NPS-2143 and KN-93 treatments effectively reduced ROS levels (Fig. 4 D). Excessive calcium uptake by mitochondria, particularly in neurons, can disrupt normal cell function by affecting ATP production, synaptic transmission, and neuronal health. In cases of sustained calcium overload, it can lead to synaptic dysfunction and promote neurodegenerative processes by triggering mitochondrial stress and cell death pathways [ , ] . Our study revealed a positive correlation between intracellular Ca 2+ levels and mitochondrial damage. In the OGD and ED groups, mitochondrial damage was exacerbated by substantial Ca 2+ influx, further intensified by the activation of the CaSR, which led to even greater Ca 2+ inflow. Mitochondria, critical for energy conversion, ATP production, and the regulation of Ca 2+ homeostasis and redox states [ ] , are abundant in brain tissue due to its high energy demands, making them essential for the normal function of nerve cells. The extent of mitochondrial damage during brain ischemia-reperfusion injury is a key determinant of brain injury severity. During cerebral ischemia-reperfusion, excessive Ca 2+ accumulates in nerve cells, and mitochondria respond by sequestering this surplus Ca 2+ from the cytoplasm, leading to mitochondrial Ca 2+ overload [ , ] . This overload is closely linked to epilepsy; recurrent seizures can cause mitochondrial damage, which disrupts Ca 2+ regulation, increases nerve excitability, and perpetuates a vicious cycle of seizures [ , ] . Furthermore, mitochondria are principal sources of ROS, which play roles in normal physiological functions. However, our findings indicated more severe mitochondrial damage and increased ROS production in the OGD&ED group. The use of CaSR agonists was associated with higher ROS levels, while CaSR and CaMKⅡ inhibitors reduced intracellular ROS. This increase in ROS during brain injury, particularly at the reperfusion stage, is due to the overactivation of enzymes and pumps hampered by ATP deficiency from ischemia and an inadequate cellular antioxidant capacity, leading to excessive ROS accumulation and mitochondrial dysfunction [ , ] . The resulting oxidative stress cascade damages mitochondrial and nuclear DNA, promotes the opening of the mitochondrial permeability transition pore, releases apoptosis-inducing factors, and triggers cell death. Additionally, cell and organelle membranes, rich in polyunsaturated fatty acids, are susceptible to ROS-induced damage and lipid peroxidation, increasing membrane permeability and Ca 2+ influx, which further leads to mitochondrial expansion and vacuolation [ ] . 3.4 Apoptosis and expression of related proteins Compared to the normal group, the apoptosis rate of HT22 cells in the OGD&ED group significantly increased, surpassing rates observed in both the ED and OGD groups. Following R568 treatment in the OGD&ED group, the apoptosis rate further escalated, whereas the application of NPS-2143 and KN-93 ameliorated the apoptosis rates (Fig. 5 A). These results align with the observed trends in intracellular Ca 2+ and its associated protein expression, increased mitochondrial damage, and heightened ROS production, indicating that Ca 2+ may play a role in apoptosis via the mitochondrial pathway in OGD&ED models. Western blot analysis revealed that the expression trends of Bax/Bcl2 and Caspase 3 were consistent with the apoptosis rates (Fig. 5 B). However, after R568 application in the OGD&ED context, Bax/Bcl2 expression did not significantly increase, while Caspase 3 expression further decreased. Immunofluorescence findings indicated that the expression trend of Bcl2 (an anti-apoptotic protein) was inversely related to the apoptosis rate (Fig. 5 C), whereas Caspase 3 expression mirrored the apoptosis rate trend (Fig. 5 C). The pro-apoptotic effect of Ca 2+ overload through mitochondrial pathway. The key to cell apoptosis is the formation of DNA fragments, and apoptosis involves the activation of DNA endonuclease in cells, leading to DNA fragmentation [ ] . In this study, TUNEL was used to detect the apoptosis of each group, based on the detection of dUTP-labeled 3'-OH termini by terminal deoxynucleotidyl transferase. The results of this study showed that Ca 2+ -overload could significantly increase the apoptosis rate of cells. Both Western blot and immunofluorescence results indicated that Bax and Bcl2 proteins were more expressed in the group with higher Ca 2+ and ROS content and more severe mitochondrial damage (the OGD&ED group). ROS accumulation and Ca 2+ -overload in mitochondria can lead to the development of permeability transformation pores and increase mitochondrial permeability, facilitating the entry of pro-apoptotic Bcl2 family proteins Bax and Bak into mitochondria, and the release of cytochrome C into the cytoplasm to activate apoptosis in the mitochondrial pathway [ ] . Bcl2 can inhibit neuronal apoptosis in cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of mitochondrial membrane permeability, direct antioxidant effects, and inhibition of intracellular calcium overload [ ] . Our results also showed that the expression of Caspase 3 increased in the OGD&ED group and decreased after the application of inhibitors, as Ca 2+ can also mediate endogenous apoptosis through the activation of Caspase 3. In cerebral ischemia-reperfusion injury, the expression ratio of anti-apoptotic protein Bcl2 to pro-apoptotic protein Bax determines whether cells undergo apoptosis. A decrease in the Bcl2/Bax ratio correlates with an increasing trend of apoptosis. Upregulation of Bcl2 and inhibition of Bax and Caspase 3 can inhibit apoptosis of nerve cells. The mechanism pathway diagram is is as follows: In conclusion, the results of this study indicate that the optimal intervention time for the OGD&ED model is 3 hours of OGD, 3 hours Mg 2+ -free, and 8 hours of reoxygenation. What’s more, our study determined that the combination of OGD and ED causes Ca 2+ overload and increased expression of Ca 2+ related proteins, and the results suggest that Ca 2+ overload is involved in regulating cell death in the mitochondrial pathway in the OGD&ED model, which will lay the foundation for research under OGD&ED conditions. Abbreviations Abbreviation Full spelling CaMKⅡ Calmodulin dependent protein kinase CaSR Calcium sensing receptor DAPI 4’,6-Diamidino-2-phenylindole dihydrochloride DMEM Dulbecco's Modified Eagle Medium ED Epileptic discharge IF Immunofluorescence ROS Reactive oxygen species OGD&ED Oxygen-glucose deprivation combined with epileptiform discharge OGD/R Oxygen-glucose deprivation / Reoxygenation PISE Post-ischemic stroke epilepsy qRT-PCR Real-time Quantitative PCR WB Western blotting Declarations Consent to Publication All authors agree to publish the article. Conflict of interest statement The authors declare no conflicts of interest. Ethical Approval and consent to participate Not applicable Availability of data and materials All data generated or analysed during this study are included in the published papers and the link https://data.mendeley.com/datasets/5699hyhccj/1.. Acknowledgment We thank Prof. Zhou for critical review of the manuscript and Prof. Qi for assistance with flow cytometry. Author contribution: Chao Gong, Jin Guo, and Shaobo Zhou contributed to the study conception and design. Material preparation, experiments, and data collection and analysis were performed by Chao Gong, Beibei Lian, Pei Zeng, Jiahao Liu, Jiawei Li, Yuanyuan Liu, and Liya Fang. The first draft of the manuscript was written by Chao Gong, Shaobo Zhou and Jin Guo, all authors commented on previous versions of the manuscript. Xunzhong Qi, Luchuan Wang and Jin Guo provided financial support. All authors read and approved the final manuscript. Funding The National Natural Science Foundation of China (No. 81300122), Fund Basic Scientific Research Operating Expenses of Provincial Institutions of Higher Learning in Heilongjiang Province (No. 2021-KYYWF-0609, No. 2022-KYYWF-0653, and 2024-KYYWF-0611) supported this study, Jiamusi University East Pole Academic Team' Children's Intelligent Rehabilitation Team (No. DJXSTD202413) and Natural Science Foundation of Heilongjiang (No.PL2024H014). References The Definition and Classification of Cerebral Palsy. Dev Med Child Neurol. 2007 Feb;49(s109):1-44. doi: 10.1111/j.1469-8749.2007.00001.x. PMID: 17371509. McIntyre S, Goldsmith S, Webb A, Ehlinger V, Hollung SJ, McConnell K, Arnaud C, Smithers-Sheedy H, Oskoui M, Khandaker G, Himmelmann K; Global CP Prevalence Group*. Global prevalence of cerebral palsy: A systematic analysis. Dev Med Child Neurol, 2022, 64(12): 1494-1506. Gong C, Liu A, Lian B, Wu X, Zeng P, Hao C, Wang B, Jiang Z, Pang W, Guo J, Zhou S. Prevalence and related factors of epilepsy in children and adolescents with cerebral palsy: a systematic review and meta-analysis. Front Pediatr, 2023, 11: 1189648. Zelnik N, Konopnicki M, Bennett-Back O, Castel-Deutsch T, Tirosh E. Risk factors for epilepsy in children with cerebral palsy. Eur J Paediatr Neurol. 2010 Jan;14(1):67-72. Gong C, Liu XP, Fang LY, Liu A, Lian BB, Qi XZ, et al. Prevalence of cerebral palsy comorbidities in China: a systematic review and meta-analysis. Front neurol, 2023, 14:1233700. Holtkamp M，Beghi E，Benninger F，et al． European stroke organisation guidelines for the management of post - stroke seizures and epilepsy． Eur Stroke J，2017，2(2): 103-115 Oumerzouk J, Vascular Epilepsy. Adv Neurol Neurosci，2021，4(2) : 5-11 owska K, Tymianski M. Calcium, ischemia and excitotoxicity. Cell Calcium, 2010, 47(2): 122-129. doi: 10.1016/j.ceca.2010.01.003. g YC. Mitochondrial dysfunction and oxidative stress in seizure-induced neuronal cell death. Acta Neurol Taiwan, 2010, 19(1): 3-15. Walters GC, Usachev YM. Mitochondrial calcium cycling in neuronal function and neurodegeneration. Front Cell Dev Biol, 2023, 11: 1094356. Sarfo FS, Akassi J, Obese V, Adamu S, Agbenorku M, Ovbiagele B. Prevalence and predictors of post-stroke epilepsy among Ghanaian stroke survivors. J Neurol Sci, 2020, 418: 117138. Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, Engel J, French J, Glauser TA, Mathern GW, Moshé SL, Nordli D, Plouin P, Scheffer IE. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia, 2010, 51(4): 676-685. Pang W, Lu S, Zheng R, Li X, Yang S, Feng Y, Wang S, Guo J, Zhou S. Investigation into Antiepileptic Effect of Ganoderic Acid A and Its Mechanism in Seizure Rats Induced by Pentylenetetrazole. Biomed Res Int, 2022, 2022: 5940372. Ryou MG, Mallet RT. An In Vitro Oxygen-Glucose Deprivation Model for Studying Ischemia-Reperfusion Injury of Neuronal Cells. Methods Mol Biol. 2018;1717:229-235. doi: 10.1007/978-1-4939-7526-6_18. PMID: 29468596. Tagin M, Shah PS, Lee KS. Magnesium for newborns with hypoxic-ischemic encephalopathy: a systematic review and meta-analysis. Journal of Perinatology, 2013, 33(9): 663-669. Fiest KM, Sauro KM, Wiebe S, Patten SB, Kwon CS, Dykeman J, Pringsheim T, Lorenzetti DL, Jetté N. Prevalence and incidence of epilepsy: A systematic review and meta-analysis of international studies. Neurology, 2017, 88(3): 296-303. Coultrap SJ, Vest RS, Ashpole NM, Hudmon A, Bayer KU. CaMKII in cerebral ischemia. Acta Pharmacol Sin, 2011, 32(7): 861-8 Toussaint F, Charbel C, Allen BG, Ledoux J. Vascular CaMKII: heart and brain in your arteries. Am J Physiol Cell Physiol. 2016, 311(3): C462–C78. De Baaij JHF, Hoenderop JGJ, Bindels RJM. Magnesium in Man: Implications for Health and Disease. Physiological Reviews, 2017, 95(1): 41-46. Thompson MD, Percy ME, Cole DEC, Bichet DG, Hauser AS, Gorvin CM. G protein-coupled receptor (GPCR) gene variants and human genetic disease. Crit Rev Clin Lab Sci. 2024 Aug;61(5):317-346. doi: 10.1080/10408363.2023.2286606. Epub 2024 Mar 18. PMID: 38497103. Wang J, Xu X, Jia W, Zhao D, Boczek T, Gao Q, Wang Q, Fu Y, He M, Shi R, Tong X, Li M, Tong Y, Min D, Wang W, Guo F. Calcium-/Calmodulin-Dependent Protein Kinase II (CaMKII) Inhibition Induces Learning and Memory Impairment and Apoptosis. Oxid Med Cell Longev. 2021 Dec 23;2021:4635054. doi: 10.1155/2021/4635054. PMID: 34976299; PMCID: PMC8718318. Dong Z, Peng Q, Pan K, Lin W, Wang Y. Microglial and Neuronal Cell Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation Aggravates Cell Injury via Activation of the Caspase-1/GSDMD Signaling Pathway. Neurochem Res, 2023, 48(9): 2660-2673. Qu Y, Liu Y, Zhang H. ALDH2 activation attenuates oxygen-glucose deprivation /reoxygenation-induced cell apoptosis, pyroptosis, ferroptosis and autophagy. Clin Transl Oncol, 2023, 25(11): 3203-3216. Li YH, Zhang S, Tang L, Feng J, Jia J, Chen Y, Liu L, Zhou J. The Role of LincRNA-EPS/Sirt1/Autophagy Pathway in the Neuroprotection Process by Hydrogen against OGD/R-Induced Hippocampal HT22 Cells Injury. J Pers Med, 2023, 13(4): 631. Wang J, Xu J, Dong Y, Su Z, Su H, Cheng Q, Liu X. ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion. FASEB J, 2023, 37(2): e22788. Zhou S, Wang SQ, Sun CY, Mao HY, Di WH, Ma XR, Liu L, Liu JX, Wang FF, Kelly P, Sreenivasaprasad P. Investigation into anti-epileptic effect and mechanisms of Ganoderma lucidum polysaccharides in in vivo and in vitro models. Proceedings of the Nutrition Society, 2015, 74(OCE1): E65. Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol, 2014, 115: 157-188. Gorini A, Villa RF. Effect of in vivo treatment of clonidine on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex. Neurochem Res, 2001, 26(7): 821-827. Lauritzen M, Mathiesen C, Schaefer K, Thomsen KJ. Neuronal inhibition and excitation, and the dichotomic control of brain hemodynamic and oxygen responses. Neuroimage, 2012, 62(2): 1040-1050. Hu HJ, Song M. Disrupted Ionic Homeostasis in Ischemic Stroke and New Therapeutic Targets. J Stroke Cerebrovasc Dis, 2017, 26(12): 2706-2719. Radak D, Katsiki N, Resanovic I, Jovanovic A, Sudar-Milovanovic E, Zafirovic S, Mousad SA, Isenovic ER. Apoptosis and Acute Brain Ischemia in Ischemic Stroke. Curr Vasc Pharmacol, 2017, 15(2): 115-122. Shutov LP, Kim MS, Houlihan PR, Medvedeva YV, Usachev YM. Mitochondria and plasma membrane Ca 2+ -ATPase control presynaptic Ca 2+ clearance in capsaicin-sensitive rat sensory neurons. J Physiol. 2013, 591(10): 2443-2462. Takemiya T, Yamagata K. Intercellular signaling pathway among Endothelia, astrocytes and neurons in excitatory neuronal damage. Int J Mol Sci, 2013, 14(4):8345-8357. Pei Z, Lee KC, Khan A, Erisnor G, Wang HY. Pathway analysis of glutamate-mediated, calcium-related signaling in glioma progression. Biochem Pharmacol, 2020, 176: 113814. Valero T. Mitochondrial biogenesis: pharmacological approaches. Curr Pharm Des, 2014, 20(35): 5507-5509. FU C Y, CHEN S J, CAI N H, et al. Increased risk of post-stroke epilepsy in Chinese patients with a TRPM6 polymorphism[J]. Neurol Res, 2019, 41(4): 378-383. Wang SQ, Li XJ, Qiu HB, Jiang ZM, Simon M, Ma XR, Liu L, Liu JX, Wang FF, Liang YF, Wu JM, Di WH, Zhou S. Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII α in epileptic hippocampal neurons. PLoS One, 2014, 9(7): e102161 Zhang L, Cao S, Deng S, Yao G, Yu T. Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor. PeerJ, 2016, 4: e2612. Zhen Y, Ding C, Sun J, Wang Y, Li S, Dong L. Activation of the calcium-sensing receptor promotes apoptosis by modulating the JNK/p38 MAPK pathway in focal cerebral ischemia-reperfusion in mice. Am J Transl Res, 2016, 8(2): 911-921. Lu FH, Tian Z, Zhang WH, Zhao YJ, Li HL, Ren H, Zheng HS, Liu C, Hu GX, Tian Y, Yang BF, Wang R, Xu CQ. Calcium-sensing receptors regulate cardiomyocyte Ca 2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation. J Biomed Sci, 2010, 17(1): 50. Takemoto-Kimura S, Suzuki K, Horigane SI, Kamijo S, Inoue M, Sakamoto M, Fujii H, Bito H. Calmodulin kinases: essential regulators in health and disease. J Neurochem, 2017, 141(6): 808-818. Robison AJ. Emerging role of CaMKII in neuropsychiatric disease. Trends Neurosci, 2014, 37(11): 653-662. Zhang X, Connelly J, Levitan ES, Sun D, Wang JQ. Calcium/Calmodulin-Dependent Protein Kinase II in Cerebrovascular Diseases. Transl Stroke Res, 2021, 12(4): 513-529. Chen XH, Chen DT, Huang XM, Chen YH, Pan JH, Zheng XC, Zeng WA. Dexmedetomidine Protects Against Chemical Hypoxia-Induced Neurotoxicity in Differentiated PC12 Cells Via Inhibition of NADPH Oxidase 2-Mediated Oxidative Stress. Neurotox Res, 2019, 35(1):139-149. Zhang X, Hong S, Qi S, Liu W, Zhang X, Shi Z, Chen W, Zhao M, Yin X. NLRP3 Inflammasome Is Involved in Calcium-Sensing Receptor-Induced Aortic Remodeling in SHRs. Mediators Inflamm, 2019, 2019: 6847087. Zhou K, Enkhjargal B, Xie Z, Sun C, Wu L, Malaguit J, Chen S, Tang J, Zhang J, Zhang JH. Dihydrolipoic Acid Inhibits Lysosomal Rupture and NLRP3 Through Lysosome-Associated Membrane Protein-1/Calcium/Calmodulin-Dependent Protein Kinase II/TAK1 Pathways After Subarachnoid Hemorrhage in Rat. Stroke, 2018, 49(1): 175-183. Ureshino RP, Erustes AG, Bassani TB, Wachilewski P, Guarache GC, Nascimento AC, Costa AJ, Smaili SS, Pereira GJDS. The Interplay between Ca 2+ Signaling Pathways and Neurodegeneration. Int J Mol Sci, 2019, 20(23): 6004. doi: 10.3390/ijms20236004. Brini M, Calì T, Ottolini D, Carafoli E. Neuronal calcium signaling: function and dysfunction. Cell Mol Life Sci, 2014, 71(15): 2787-2814. doi: 10.1007/s00018-013-1550-7. Verma M, Lizama BN, Chu CT. Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration. Transl Neurodegener, 2022, 11(1): 3. Zhao S, Feng H, Jiang D, Yang K, Wang ST, Zhang YX, Wang Y, Liu H, Guo C, Tang TS. ER Ca 2+ overload activates the IRE1α signaling and promotes cell survival. Cell Biosci, 2023, 13(1): 123. Wang CH, Wei YH. Role of mitochondrial dysfunction and dysregulation of Ca 2+ homeostasis in the pathophysiology of insulin resistance and type 2 diabetes. J Biomed Sci, 2017, 24(1): 70. Wu M, Gu X, Ma Z. Mitochondrial Quality Control in Cerebral Ischemia-Reperfusion Injury. Mol Neurobiol. 2021, 58(10): 5253-5271. Jia J, Jin H, Nan D, Yu W, Huang Y. New insights into targeting mitochondria in ischemic injury. Apoptosis, 2021, 26(3-4): 163-183. Sumadewi KT, Harkitasari S, Tjandra DC. Biomolecular mechanisms of epileptic seizures and epilepsy: a review. Acta Epileptologica 5, 2023, 28. Madireddy S, Madireddy S. Therapeutic Strategies to Ameliorate Neuronal Damage in Epilepsy by Regulating Oxidative Stress, Mitochondrial Dysfunction, and Neuroinflammation. Brain Sci, 13(5): 784. Sanderson TH, Reynolds CA, Kumar R, Przyklenk K, Hüttemann M. Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol Neurobiol, 2013, 47(1): 9-23. Wu L, Xiong X, Wu X, Ye Y, Jian Z, Zhi Z, Gu L. Targeting Oxidative Stress and Inflammation to Prevent Ischemia-Reperfusion Injury. Front Mol Neurosci, 2020, 13: 28. Endale HT, Tesfaye W, Mengstie TA. ROS induced lipid peroxidation and their role in ferroptosis. Front Cell Dev Biol, 2023, 11: 1226044. Coletti C, Acosta GF, Keslacy S, Coletti D. Exercise-mediated reinnervation of skeletal muscle in elderly people: An update. Eur J Transl Myol, 2022, 32(1): 10416. Yoshida A, Pommier Y, Ueda T. Endonuclease activation and chromosomal DNA fragmentation during apoptosis in leukemia cells. Int J Hematol. 2006 Jul;84(1):31-37. Zhang L, Li D, Yin L, Zhang C, Qu H, Xu J. Neuroglobin protects against cerebral ischemia/reperfusion injury in rats by suppressing mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal apoptosis through synaptotagmin-1. Environ Toxicol, 2023, 38(8): 1891-1904. Additional Declarations No competing interests reported. Supplementary Files BTheexpressionofCaSRandCaMKIIwasdetectedbyWB.zip BTheexpressionofCasepase3andBcl2proteinwasdetectedbyWB.zip fulluncroppedGelsandBlotsimages1.zip fulluncroppedGelsandBlotsimages2.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 21 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviews received at journal 17 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 03 Jul, 2025 Editor invited by journal 29 May, 2025 Editor assigned by journal 26 May, 2025 Submission checks completed at journal 26 May, 2025 First submitted to journal 13 May, 2025 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-6659867","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":480769174,"identity":"ab63bcc8-40d6-42a9-9ebb-449acb3c9e19","order_by":0,"name":"Chao Gong","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Gong","suffix":""},{"id":480769175,"identity":"e8d2c456-ba99-4f97-8167-c098466ebd13","order_by":1,"name":"Xunzhong Qi","email":"","orcid":"","institution":"Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Xunzhong","middleName":"","lastName":"Qi","suffix":""},{"id":480769176,"identity":"a4bdb6c6-a5cb-49f6-b4d4-a8014cdf569f","order_by":2,"name":"Luchuan Wang","email":"","orcid":"","institution":"Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Luchuan","middleName":"","lastName":"Wang","suffix":""},{"id":480769177,"identity":"5af19a02-725e-4e80-9196-a9ac43857c48","order_by":3,"name":"Pei Zeng","email":"","orcid":"","institution":"Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Zeng","suffix":""},{"id":480769178,"identity":"ca58bdbb-359b-4790-9999-2f4b6387d39b","order_by":4,"name":"Beibei Lian","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Beibei","middleName":"","lastName":"Lian","suffix":""},{"id":480769179,"identity":"69d362a1-bd5f-4df3-9177-890a04998259","order_by":5,"name":"Jiahao Liu","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Jiahao","middleName":"","lastName":"Liu","suffix":""},{"id":480769180,"identity":"1ed3d7ed-3a21-4956-a4af-63917ab9c062","order_by":6,"name":"Jiawei Li","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Li","suffix":""},{"id":480769181,"identity":"74d0d7c2-6ab6-4c58-880b-b6ad26fd906d","order_by":7,"name":"Liya Fang","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Liya","middleName":"","lastName":"Fang","suffix":""},{"id":480769182,"identity":"4c19ed9e-5503-483c-81db-964dbd48fb34","order_by":8,"name":"Yuanyuan Liu","email":"","orcid":"","institution":"Third Affiliated Hospital of Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Liu","suffix":""},{"id":480769183,"identity":"d688b563-5d98-4765-b973-921c1bd46f9a","order_by":9,"name":"Jin Guo","email":"","orcid":"","institution":"Jiamusi University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Guo","suffix":""},{"id":480769184,"identity":"4bf1b151-0373-49b5-9f59-3c9653cc0433","order_by":10,"name":"Shaobo Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYHACNiC2gbINGBIgjAMEtaSRruUwnEdYi3wD+7MHH9vO221vbz72mKfALo+B/fADZp4zuLUYHGBIN5zZdjt5zplj6cY8BsnFDDxpBsw8N/BoYWA4Js0L1CIhkWMmnWNwILGBIYeBmecDPocxtgG1nEuWkH//DaKF/w1+LQwHmNmAWg7YSUjwsEG0SIBsweeww2xskjPOJSdI8KSZSf8xSE5sk3hmcHAOHu/Lt7c/k/hQZmcvwX74meSMP3aJ/fzJDx+8OYbHYcwQCuhrKABF0wE8GuDAnhhFo2AUjIJRMEIBAAN4SlN/MAIjAAAAAElFTkSuQmCC","orcid":"","institution":"University of Greenwich","correspondingAuthor":true,"prefix":"","firstName":"Shaobo","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-05-14 03:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6659867/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6659867/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86145323,"identity":"c39cf89b-b146-4c30-8f56-f0e16ea4b270","added_by":"auto","created_at":"2025-07-07 09:03:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1182838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe experimental designflow chart. Notes\u003c/strong\u003e: CaMKⅡ:Calmodulin dependent protein kinase; CaSR: Calcium sensing receptor; IF: Immunofluorescence; ROS: Reactive oxygen species; OGD: Oxygen-glucose deprivation; R: Reoxygenation; qRT-PCR: Real-time Quantitative PCR; WB: Western blotting.\u003c/p\u003e","description":"","filename":"fig.1flowchart.png","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/f4127e39484c025ecc4dd3b1.png"},{"id":86146337,"identity":"9f3e0a93-b9be-4609-9213-48a6f293af58","added_by":"auto","created_at":"2025-07-07 09:11:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2164688,"visible":true,"origin":"","legend":"\u003cp\u003eImpacts of varied oxygen-glucose deprivation (OGD), Mg\u003csup\u003e2+\u003c/sup\u003e-free conditions, and reoxygenation durations on cell viability. (A) Cell viability under different OGD, magnesium-free and oxygen recovery time, n=3. (B) The degree of cell viability decline after magnesium-free 6h. B-a) Different OGD time. The decline in cell viability is most obvious when OGD=3 hours; B-b) Different reoxygenation time. The decline in cell viability is most obvious when the reoxygenation=8 hours. (C and D) The effects of different magnesium-free time on intracellular Ca\u003csup\u003e2+\u003c/sup\u003e When OGD=3h and reoxygenation=8h. The decline in cell viability is most obvious when the magnesium-free=3 hours (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/4e6a87e2a8cffbc950f57843.png"},{"id":86146339,"identity":"0543b6b8-7c5f-4541-9558-0c2e4408b45e","added_by":"auto","created_at":"2025-07-07 09:11:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551945,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e-related proteins in each group. (A) Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e in each group. Compared with the control group, it was significantly increased in the OGD\u0026amp;ED group (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e=0.003). Compared with the OGD\u0026amp;ED group, it was further increased after R568 intervention (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), but it was significantly decreased after NPS-2143 and KN-93 intervention (\u003csup\u003e++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Scale=50μm, n=3. (B) The expression of CaSR and CaMKⅡ protein in each group was detected by WB. Compared with the normal group, CaSR protein was increased in the OGD\u0026amp;ED group (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). Compared with the OGD\u0026amp;ED group, CaSR protein was significantly decreased in the OGD\u0026amp;ED+NPS-2143 group (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the normal group, CaMKⅡ protein was significantly increased in the OGD\u0026amp;ED group (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the OGD\u0026amp;ED group,CaMKⅡ protein was decreased in the OGD\u0026amp;ED+KN-93 group (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), n=5. (C) The expression of CaSR and CaMKⅡ was detected by IF (a.Normal, b.ED, c.OGD, d.OGD\u0026amp;ED, e.OGD\u0026amp;ED+R568, f.OGD\u0026amp;ED+NPS-2143, g.OGD\u0026amp;ED+KN-93). Compared with normal group, CaSR protein in ED, OGD and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the OGD\u0026amp;ED group, CaSR protein in the OGD\u0026amp;ED+R568 group was significantly increased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), but CaSR protein in the OGD\u0026amp;ED+NPS-2143 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the normal group, CaMKⅡ protein in OGD and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with OGD\u0026amp;ED group, CaMKⅡ protein in OGD\u0026amp;ED+KN-93 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Scale =100μm, n=3. (D) mRNA expression of CaSR and CaMKⅡ was detected by qRT-PCR. Compared with the normal group, the mRNA expression of CaSR in the OGD and the OGD\u0026amp;ED group was significantly increased (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). Compared with the OGD\u0026amp;ED group, CaSR in the OGD\u0026amp;ED+R568 group was significantly increased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), while was significantly decreased in the OGD\u0026amp;ED+NPS-2143 group(\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Compared with the normal group, the mRNA expression of CaMKⅡ in the OGD and the OGD\u0026amp;ED group was increased (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). Compared with the OGD\u0026amp;ED group, CaMKⅡ in the OGD\u0026amp;ED+KN-93 group was significantly decreased (\u003csup\u003e+++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e=0.002), n=3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/4a5db64178620e47e6c3b4a0.png"},{"id":86145332,"identity":"6f6b6a92-351a-4607-84a1-14089f5e5fc8","added_by":"auto","created_at":"2025-07-07 09:03:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5630221,"visible":true,"origin":"","legend":"\u003cp\u003eCell status, viability, and mitochondria of each group. (A) Cell status. Compared with the Normal group, hippocampal neurons in the ED and OGD groups had shrunken and collapsed somata, thinned, atrophied, broken and necrotic neurites, weakened wall - adhering ability and detachment, while compared with the OGD\u0026amp;ED group, R568 intervention during reoxygenation aggravated cell damage, and NPS-2143 and KN-93 interventions improved HT22 cell morphology, increased adherent cell number and enhanced cell growth state. Scale =100μm. (B) CCK-8 was used to detect cell viability in each group. Compared with the OGD\u0026amp;ED group, the cell viability of the OGD\u0026amp;ED+R568 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), but the cell viability of the OGD\u0026amp;ED+NPS-2143 and the OGD\u0026amp;ED+KN-93 groups were improved (\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), n=3. (C) The mitochondrial status of each group was observed by transmission electron microscopy (TEM). Obvious mitochondrial lysis and ballooning degeneration occurred in the OGD\u0026amp;ED group. Compared with the OGD\u0026amp;ED group, after the application of R568, the mitochondrial damage was more severe; after the application of NPS-2143 and KN-93, the mitochondrial lysis and ballooning degeneration were reduced. Scale =1µm. (D) Intracellular ROS levels. Compared with the normal group, ROS in ED, OGD and OGD\u0026amp;ED groups were increased (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05). Compared with the OGD\u0026amp;ED group, ROS was significantly increased after R568 intervention (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001) and significantly decreased after NPS-2143 and KN-93 intervention (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Scale =100μm, n=3.\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/1e92e2b469aa12f55fa6f384.png"},{"id":86145336,"identity":"9962552f-0700-4570-a042-d0394fda4755","added_by":"auto","created_at":"2025-07-07 09:03:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":765799,"visible":true,"origin":"","legend":"\u003cp\u003eCell apoptosis rate and expression of apoptosis-related proteins in each group. (A) Cell apoptosis rate in each group by tunel. Compared with the normal group, the apoptosis rate in ED, OGD, and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the OGD\u0026amp;ED group, R568 could further increase the apoptosis rate (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), and NPS-2143 and KN-93 could improve the apoptosis rate (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Scale =100μm, n=3. (B) The expression of apoptosis-related proteins was detected by WB. Compared with the normal group, the expression of Bax protein in ED, OGD, and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). Bax protein in the OGD\u0026amp;ED+ NPS-2143 group and the OGD\u0026amp;ED+KN-93 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). The expression of Bcl2 protein was significantly decreased in ED, OGD, and OGD\u0026amp;ED groups compared with the normal group (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). The ratio of Bax/Bcl2 in ED, OGD, and OGD\u0026amp;ED groups was significantly higher than in the normal group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005). \u0026nbsp;The Bcl2 protein in OGD\u0026amp;ED+NPS-2143 and OGD\u0026amp;ED+KN-93 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Compared with the normal group, the expression of Caspase 3 protein in OGD and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Caspase 3 protein was significantly decreased in the OGD\u0026amp;ED+NPS-2143 group and the OGD\u0026amp;ED+KN-93 group(\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), n=5. (C) The expression of apoptosis-related proteins was detected by IF(a. Normal, b.ED, c.OGD, d.OGD\u0026amp;ED, e.OGD\u0026amp;ED+R568, f.OGD\u0026amp;ED+NPS-2143, g.OGD\u0026amp;ED+KN-93). Compared with the normal group, the fluorescence of Bcl2 protein in ED, OGD, and OGD\u0026amp;ED groups was significantly decreased (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the OGD\u0026amp;ED group, Bcl2 protein in the OGD\u0026amp;ED+R568 group was significantly decreased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), and Bcl2 protein in OGD\u0026amp;ED+NPS-2143 and OGD\u0026amp;ED+KN-93 groups was significantly increased (\u003csup\u003e++++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the normal group, the fluorescence intensity of Caspase 3 protein in ED, OGD, and OGD\u0026amp;ED groups was significantly increased (\u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). Compared with the OGD\u0026amp;ED group, Caspase 3 protein in the OGD\u0026amp;ED+R568 group was enhanced (\u003csup\u003e+\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e=0.025), but it was significantly decreased (\u003csup\u003e+++\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.005) in the OGD\u0026amp;ED+NPS-2143 and the OGD\u0026amp;ED+KN-93 group. Scale=100μm, n=3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/12b6baf3a18c0fe5a1ef0876.png"},{"id":86146877,"identity":"f8a8bdc8-9919-4575-b7a2-28415f9861c9","added_by":"auto","created_at":"2025-07-07 09:19:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":113463,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the \u003cstrong\u003eResults and discussion \u003c/strong\u003esection.\u003c/p\u003e","description":"","filename":"Mechanismdiagram.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/63ee1fcc2443d590f24f66f1.jpg"},{"id":86146880,"identity":"9609efb3-942d-43f0-bddc-0ce339afa2d1","added_by":"auto","created_at":"2025-07-07 09:19:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11470982,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/2f9cb56d-9fbb-4e22-9015-1d9d80752294.pdf"},{"id":86145327,"identity":"06c5df80-d045-4ebf-be2e-3def4c140abc","added_by":"auto","created_at":"2025-07-07 09:03:19","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12348382,"visible":true,"origin":"","legend":"","description":"","filename":"BTheexpressionofCaSRandCaMKIIwasdetectedbyWB.zip","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/9032c185a45da75cc3170892.zip"},{"id":86145324,"identity":"b96e6e2e-bd5d-4c7c-bb33-775832fba70b","added_by":"auto","created_at":"2025-07-07 09:03:19","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14465205,"visible":true,"origin":"","legend":"","description":"","filename":"BTheexpressionofCasepase3andBcl2proteinwasdetectedbyWB.zip","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/fcfffb40e93ea6b61748f48a.zip"},{"id":86145351,"identity":"cf06812a-31e2-4ebe-b0fe-b1ca43f1f25b","added_by":"auto","created_at":"2025-07-07 09:03:20","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12348382,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages1.zip","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/c711c7b71195dc1fc403f6d7.zip"},{"id":86145348,"identity":"7446ea89-4475-4cb1-907e-f88a98b71f60","added_by":"auto","created_at":"2025-07-07 09:03:20","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14465205,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages2.zip","url":"https://assets-eu.researchsquare.com/files/rs-6659867/v1/60fc86bbcb8e71ef8d626847.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCerebral palsy (CP) is a group of lifelong central motor disorder syndromes that significantly affect early childhood survival and quality of life\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn1\" id=\"#FNLinkFn1\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. According to population-based studies, the global prevalence of CP ranges from 0.1\u0026ndash;0.4%, making it one of the most common conditions threatening early childhood survival\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn2\" id=\"#FNLinkFn2\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Epilepsy is a common clinical manifestation in children with CP and is widely regarded as an indicator of CP severity. While the prevalence of epilepsy in the general population is estimated to be around 0.4\u0026ndash;0.8%, it is notably higher among children with CP, with research from our group suggesting a prevalence ranging from 31.6\u0026ndash;46.5%\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn3\" id=\"#FNLinkFn3\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. This co-occurrence may be attributed to shared pathophysiological mechanisms associated with common etiologies and risk factors, resulting in the simultaneous onset of both CP and epilepsy\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn4\" id=\"#FNLinkFn4\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn5\" id=\"#FNLinkFn5\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Post-ischemic stroke epilepsy (PISE) refers to non-provoked epileptic seizures that occur two or more times (with an interval of 24 hours) in patients with no previous history of epilepsy, and these seizures are secondary to ischemic brain tissue damage caused by severe stenosis or occlusion of blood vessels\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn6\" id=\"#FNLinkFn6\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Ischemic stroke is a common cause of secondary epilepsy in the elderly aged over 60 worldwide, with an incidence rate as high as 25%-50%. Some studies have shown a long-term cumulative risk of PISE, with the proportion of late-onset epileptic seizures in all PISE patients being 2%-15%\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn7\" id=\"#FNLinkFn7\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Despite its prevalence, the exact mechanisms linking epilepsy to CP and stroke remain unclear. In many cases, the cause of epilepsy in children with CP and stroke is unknown. A key factor may be ischemia-reperfusion injury, which occurs after cerebral ischemia and glucose deprivation. In this scenario, the restoration of blood flow does not lead to recovery but instead results in greater dysfunction. The oxygen-glucose deprivation/reoxygenation (OGD/R) model effectively simulates this process. The epileptic discharge (ED) model, induced by incubation in Mg\u003csup\u003e2+\u003c/sup\u003e-free extracellular fluid, is a widely used in vitro model to replicate epileptic seizures. Studies have demonstrated that the absence of Mg\u003csup\u003e2+\u003c/sup\u003e leads to intracellular Ca\u003csup\u003e2+\u003c/sup\u003e influx, causing mitochondrial damage and ultimately resulting in cell death.\u003c/p\u003e \u003cp\u003eCalcium homeostasis, serving as a key second messenger, plays a crucial role in maintaining neuronal function. An increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e (often referred to as Ca\u003csup\u003e2+\u003c/sup\u003e overload) can trigger several neurotoxic mechanisms. Both cerebral ischemia-reperfusion injury and epilepsy can lead to Ca\u003csup\u003e2+\u003c/sup\u003e overload, which induces neuronal damage via mitochondrial dysfunction, activation of calcium-dependent degrading enzymes, and increased production of oxygen free radicals\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn8\" id=\"#FNLinkFn8\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn9\" id=\"#FNLinkFn9\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Mitochondria play essential roles in ATP synthesis, reactive oxygen species (ROS) production, Ca\u003csup\u003e2+\u003c/sup\u003e buffering, and apoptosis signaling. In neurons, mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e buffering is critical for maintaining neuronal excitability, synaptic transmission, and overall cell function\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn10\" id=\"#FNLinkFn10\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload is a common pathological mechanism underlying neurological conditions, including mitochondria-related epilepsy and post-stroke epilepsy.\u003c/p\u003e \u003cp\u003eEpilepsy can be caused by various neurological conditions, including stroke (11.4%\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn11\" id=\"#FNLinkFn11\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e), cerebral palsy (31.6%-46.5%\u003csup\u003e[3]\u003c/sup\u003e), and traumatic brain injury. Brain injury often leads to structural changes in neural networks and increased neuronal excitability, which heightens the risk of epilepsy\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn12\" id=\"#FNLinkFn12\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn13\" id=\"#FNLinkFn13\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Ischemia causes a deficiency in oxygen and glucose supply to the brain, and the longer the restoration of blood flow is delayed, the more severe the resulting dysfunction. This process is referred to as ischemia-reperfusion injury, which forms the basis for the development of the OGD/R model. The OGD/R model is widely used in research to simulate ischemic stroke in vitro, while the ED model, created by culturing cells in a Mg\u003csup\u003e2+\u003c/sup\u003e-free extracellular environment, is commonly used to simulate epileptic seizures\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn14\" id=\"#FNLinkFn14\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Studies have shown that a lack of intracellular Mg\u003csup\u003e2+\u003c/sup\u003e can lead to Ca\u003csup\u003e2+\u003c/sup\u003e influx, causing mitochondrial damage and subsequent cell death\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn15\" id=\"#FNLinkFn15\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the strong association between epilepsy and both CP and stroke, particularly in cases involving ischemic brain injury, there is a need for further research in this area. While the OGD/R model is commonly used in studies to investigate stroke, its application in simulating CP-related ischemic brain injury remains limited. This study aims to build on existing research by introducing a Mg\u003csup\u003e2+\u003c/sup\u003e-free intervention during the reoxygenation phase of OGD, thereby establishing an OGD\u0026amp;ED model that can serve as a reference for future investigations. Furthermore, both OGD/R and ED have been shown to cause intracellular Ca\u003csup\u003e2+\u003c/sup\u003e overload, leading to mitochondrial dysfunction and apoptosis. This study will explore the mechanisms underlying Ca\u003csup\u003e2+\u003c/sup\u003e overload in this OGD\u0026amp;ED model and provide insights into the potential therapeutic targets for mitigating neuronal damage. Epilepsy is one of the most common neurological disorders, affecting about 0.5%~1% of the world's population\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn16\" id=\"#FNLinkFn16\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Epilepsy is caused by the abnormal firing of neurons due to an imbalance of Ca\u003csup\u003e2+\u003c/sup\u003e in the neurons. Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, which includes Ca\u003csup\u003e2+\u003c/sup\u003e influx, efflux, caching, storage, and diffusion, plays a significant role in maintaining cell function and acts as a ubiquitous second messenger. The disruption of calcium homeostasis is a major initiator and activator of the cell death pathway\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn17\" id=\"#FNLinkFn17\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn18\" id=\"#FNLinkFn18\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Ca\u003csup\u003e2+\u003c/sup\u003e overload refers to the abnormal increase of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e caused by various reasons, leading to abnormal phenomena of cell structure and function damage. Both cerebral ischemia-reperfusion and epilepsy can cause Ca\u003csup\u003e2+\u003c/sup\u003e overload, causing damage to nerve cells. Research has shown that it may involve three major mechanisms: mitochondrial dysfunction, activation of calcium-dependent degrading enzymes, and promoting oxygen-free radical generation\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn19\" id=\"#FNLinkFn19\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe calcium-sensing receptor (CaSR) plays a crucial role in calcium homeostasis. Abnormal CaSR function may lead to nervous system diseases, including epilepsy\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn20\" id=\"#FNLinkFn20\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Current research indicates that abnormal changes in the calmodulin-dependent protein kinase II (CaMKII) pathway, which is associated with epileptogenesis, lead to decreased CaMKII levels in multiple epileptic models\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn21\" id=\"#FNLinkFn21\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. In this study, we first established a model to determine the optimal time for generating characteristic features of both OGD and ED conditions, then applied Ca\u003csup\u003e2+\u003c/sup\u003e regulators, including CaSR and CaMKII agonists and inhibitors, to investigate the effects of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e overload on mitochondrial dysfunction and cell apoptosis, with a focus on the pro-apoptotic effects of the mitochondrial pathway.\u003c/p\u003e"},{"header":"2. Method and materials","content":"\u003cp\u003eThe whole experiment was shown in the flow chart, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eMouse hippocampal neuron HT22 cells (RRID: CVCL_0321) were obtained from Shanghai Heisou Biotechnology, Shanghai, China. HT22 has not been listed as a commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC). Its passage number is limited to 5 times. The cell culture medium was prepared with 10% Fetal Bovine Serum (FBS, RRID: SCR_014489, Gibco, the United States) and 1% penicillin (cat. no. C0222, Beyotime, China). The Mg\u003csup\u003e2+\u003c/sup\u003e-free solution was prepared with NaCl (124.0 mmol/L), NaHCO3 (25.0 mmol/L), KCl (3.5mmol/L), CaCl2 (2.5 mmol/L), NaH2PO4 (1.2 mmol/L) and glucose (10.0 mmol/L), and the pH was adjusted to 7.4 with 1.0 mol/L HCl.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Model preparation\u003c/h2\u003e \u003cp\u003eTo explore cell viability and Ca\u003csup\u003e2+\u003c/sup\u003e concentration in OGD\u0026amp;ED model cells, HT22 cells were subjected to OGD for 1 to 6 hours, followed by Mg\u003csup\u003e2+\u003c/sup\u003e-free incubation for 0 to 6 hours, and reoxygenation for 8, 12, and 24 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (cat. no. C0038, Beyotime, China), during both OGD and reoxygenation phases. Furthermore, the Fluo-4 AM probe (cat. no. S1060, Beyotime, China) was utilized to determine the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration over a 0 to 6-hour Mg\u003csup\u003e2+\u003c/sup\u003e-free. Ca\u003csup\u003e2+\u003c/sup\u003e overload in the subsequent studies were investigated under conditions that result in the highest Ca\u003csup\u003e2+\u003c/sup\u003e concentration.\u003c/p\u003e \u003cp\u003eThere were seven groups in the experiment, they were, 1. Control group: HT22 cells were conventionally cultured with normal oxygen and Mg\u003csup\u003e2+\u003c/sup\u003e with no treatment. Briefly, when the monolayer of HT22 cells in the petri dish reaches about 80% confluence, proceed with subculturing. Discard the supernatant, wash the cells with PBS 2\u0026ndash;3 times, and add 2 mL of 0.25% trypsin solution for digestion. After 2 minutes, 1 mL of complete medium was added to stop the digestion, gently blew the adherent cells off to form a cell suspension, and transferred it to a 15 mL centrifuge tube, centrifuged at 1400 rpm for 5 minutes. The supernatant was discarded, complete medium was added to gently resuspend the centrifuged cells into a single-cell suspension, and then subcultured at a ratio of 1:3 into new culture dishes for continuous cultivation. 2. OGD group: HT22 cells in the logarithmic growth phase were quantified and seeded them into plates well and cultured in a regular medium until confluency, then the medium was switched to a sugar-free Dulbecco's Modified Eagle Medium (DMEM, cat. no. 11966025 Gibco, the United States), placed in an anaerobic tank, and incubated at 37℃ for hypoxia. After rinsing with sterile PBS, the medium was switched to a normal culture medium for reoxygenation with sugar-containing medium. 3. ED group: HT22 cells were incubated in a Mg\u003csup\u003e2+\u003c/sup\u003e-free extracellular solution until confluency, then the medium was switched to sugar-containing medium. 4. OGD\u0026amp;ED group: Upon reaching confluency, HT22 cells were cultured in sugar-free DMEM, placed in an anaerobic tank, incubated at 37℃ for hypoxia, followed by reoxygenation first with Mg\u003csup\u003e2+\u003c/sup\u003e-free extracellular solution, then with sugar-containing medium. 5. OGD\u0026amp;ED\u0026thinsp;+\u0026thinsp;R568 group: Similar to the OGD\u0026amp;ED group, but with 10 \u0026micro;mol/L CaSR agonist [R568 (cat. no. HY-10167, MCE, the United States)] added during the reoxygenation phase. 6. OGD\u0026amp;ED\u0026thinsp;+\u0026thinsp;NPS-2143 group: Based on the OGD\u0026amp;ED protocol, but with 10 \u0026micro;mol/L CaSR inhibitor [NPS-2143 (cat. no. HY-10007, MCE, the United States)] added during reoxygenation. 7. OGD\u0026amp;ED\u0026thinsp;+\u0026thinsp;KN-93 group: Following the OGD\u0026amp;ED procedure, with the addition of 10\u0026micro;mol/L CaMK II inhibitor [KN-93(cat. no. HY-15465, MCE, the United States)] during reoxygenation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell activity\u003c/h2\u003e \u003cp\u003eCell viability and proliferation were assessed using the CCK-8 assay as per manufacturer's instructions. Cells were seeded in 96-well plates at 5\u0026times;10^4 cells/well. Post-treatment, 10 \u0026micro;L of CCK-8 solution was added to each well and incubated at 37℃ for 2 hours. Absorbance was measured at 450 nm using an enzymoleter, and cell viability was calculated and plotted.\u003c/p\u003e \u003cp\u003eIntracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration measurement\u003c/p\u003e \u003cp\u003eCells were seeded onto 24-well plates and, upon reaching approximately 60% adhesion, they were rinsed with sterile PBS following the experimental protocol. Then, 250 \u0026micro;L of Fluo-4 dye solution was added to each well and incubated for 30 minutes at 37\u0026ordm;C in the dark. Post-incubation, the staining was examined under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 The morphology of hippocampal neurons\u003c/h2\u003e \u003cp\u003eAfter the intervention was performed on approximately 50% of the adherent cells, the morphological changes were observed using an inverted phase contrast microscope. Cell images were taken using a camera under a microscope for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Detection of intracellular ROS\u003c/h2\u003e \u003cp\u003eCells were seeded onto 24-well culture plates. Following the intervention, the culture medium was removed, and fresh serum-free medium with 10\u0026micro;moL/DCFH-DA probe (cat. no. S0034S, Beyotime, China) was added, then incubated at 37℃ for 20 minutes. The cells were washed three times with serum-free medium before being observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Morphology of mitochondria\u003c/h2\u003e \u003cp\u003eCells were cultured in a dish and treated at 80% confluence. HT22 cells were trypsinized, washed with PBS, and centrifuged at 800 rpm for 10 min. The supernatant was discarded, and the cell pellet fixed in 2.5% glutaraldehyde for 24 hours, followed by 1% osmium tetroxide for 1 hour, washed once with PBS, dehydrated in an ethanol series, and embedded in EPON resin. Thin sections were stained with 1% uranyl acetate and lead citrate, and the mitochondrial structure observed by transmission electron microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Apoptosis assessment\u003c/h2\u003e \u003cp\u003eAfter the intervention, cells from the seven groups were collected, the culture medium was removed, and cells were washed with PBS, fixed with immunofixation solution for 30 minutes at room temperature, washed again with PBS, and treated with 0.3% Triton X-100 in PBS for 5 minutes at room temperature, followed by 2\u0026ndash;3 PBS washes. The TUNEL (cat. no. MA0223, MeilunBio, China) reaction mixture was prepared as per the instructions, and 50 \u0026micro;L was added to each sample, covered with anti-evaporation film, and incubated in a humidified chamber at a constant temperature for 60 minutes in the dark. After incubation, cells were washed three times with PBS, stained with 4\u0026rsquo;,6-Diamidino-2-phenylindole dihydrochloride (DAPI, cat. no. MA0128, MeilunBio, China) for 5 minutes in the dark, followed by three PBS washes. Images were collected using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot assay\u003c/h2\u003e \u003cp\u003eIn this study, we presented the full membranes with all target proteins and GAPDH together. Proteins were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked in TBST containing 5% skim milk powder at room temperature for 2 hours, followed by incubation with primary antibodies at 4\u0026deg;C: CaSR (1:2000, cat. no. AB_10646434, Proteintech, the United States), CaMKII (1:1000, cat. no. ab134041, Abcam, the United Kingdom), Bax (1:2000, cat. no. AB_2819658, Proteintech, the United States), Bcl2 (1:1000, cat. no. AB_2918886, Proteintech, the United States), Caspase 3 (1:1000, cat. no. 82202-1-RR, Proteintech, the United States), and GAPDH (1:50000, cat. no. 60004-1-Ig, Proteintech, the United States). After primary antibody incubation, the membrane was incubated with HRP-conjugated secondary antibodies (1:20000, cat. no. BA1070, Boster, China) at room temperature for 2 hours. Chemiluminescence signals were visualized using a chemiluminescence system, and the intensity of the bands was quantified using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eCaSR (1:150), CaMKII (1:250), Bcl2 (1:400), and Caspase 3 (1:50) in HT22 cells were detected by immunofluorescence labeling. Each group was fixed, permeabilized, blocked, and incubated with the first antibody at 4 ℃ overnight, followed by incubation with the fluorescent goat anti-rabbit secondary antibody (cat. no. BA1091 and BA1090, Boster, China) at 37 ℃ for 1.5 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 qRT-PCR analysis\u003c/h2\u003e \u003cp\u003eCell samples were processed for qRT-PCR analysis using a cDNA kit (cat. no. RR036A and RR820A-200Rx, Takara, Japan) according to the provided guidelines. The qRT-PCR was performed on an automatic medical PCR instrument (SLAN-96P) using SYBR Green qPCR Master Mix. The reverse transcription step involved incubation at 95\u0026deg;C for 10 minutes, followed by an hour at 42\u0026deg;C. For the qPCR amplification, a Real-Time reaction mixture of 5\u0026micro;l was prepared in a total volume of 50\u0026micro;l, containing custom gene-specific primers for the target sequences. The qPCR amplification consisted of 40 cycles, with denaturation at 95\u0026deg;C for 15 seconds, followed by annealing at 56\u0026deg;C for 60 seconds. Primers for CaSR were 5\u0026prime;-ACCTTTACCTGTCCCCTGAA-3\u0026prime; (Forward) and 5\u0026prime;-GGGCAACAAAACTCAAGGTG-3\u0026prime; (Reverse); for CaMKII, 5'-GCGACCTGAAGCCTGAGAATCTG \u0026minus;\u0026thinsp;3'(Forward) and 5'-GTGTCCCTGCGAACCCAAACC-3' (Reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistics\u003c/h2\u003e \u003cp\u003eStatistical analysis and plotting were performed using GraphPadPrism8.0.2 and SPSS18.0. Each experimental group for every detection index was repeated 3 to 5 times, and results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Data were assessed for normality, using the Shapiro\u0026ndash;Wilk test. All data showed a \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for this, indicating they were normally distributed. The generalised ESD test was conducted to determine whether significant outliers existed. The test showed no outliers, and no data points were excluded from the analysis.\u003c/p\u003e \u003cp\u003eUnivariate analysis of variance and repeated measures analysis of variance were utilized for statistical analysis, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The data that support the findings of this study are openly available in Mendeley at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://data.mendeley.com/datasets/5699hyhccj/1\u003c/span\u003e\u003cspan address=\"https://data.mendeley.com/datasets/5699hyhccj/1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Exploration of the OGD\u0026amp;ED Model\u003c/h2\u003e \u003cp\u003eExtensive prior research has been dedicated to identifying optimal durations for OGD in nerve cell OGD/Reoxygenation (OGD/R) models \u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn22\" id=\"#FNLinkFn22\"\u003e\u003c/a\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn23\" id=\"#FNLinkFn23\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. A comprehensive review of the current literature revealed that the most commonly employed OGD/R durations are 2 and 4 hours, with Mg\u003csup\u003e2+\u003c/sup\u003e-free conditions frequently applied for 3 hours\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn24\" id=\"#FNLinkFn24\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. This insight underscores the importance of precisely calibrating intervention times to navigate the considerable variability observed in cellular responses across differing experimental setups and conditions.\u003c/p\u003e \u003cp\u003eThe CCK-8 assay relies on the oxidation of water-soluble tetrazolium salt-8, which is reduced by cellular dehydrogenases to form a soluble formazan dye to produce yellow coloration. This allows the measurement of cell metabolic activity by dehydrogenases in active mitochondria and allows a direct metric of cell viability. This assay is directly proportional to the count of viable cells. In this investigation, 18 specific time points were delineated across varying conditions (OGD periods from 1 to 6 hours, Mg\u003csup\u003e2+\u003c/sup\u003e-free periods from 0 to 6 hours, and reoxygenation intervals at 8, 12, and 24 hours) as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, to systematically evaluate cell viability using the CCK-8 assay.\u003c/p\u003e \u003cp\u003eInitial analyses focused on discerning the impacts of various intervention intervals within the first hour, notably identifying a significant suppression of cell viability during the initial hour of Mg\u003csup\u003e2+\u003c/sup\u003e-free intervention. This observation hints at the potential of short-term Mg\u003csup\u003e2+\u003c/sup\u003e-free conditions during reoxygenation to induce epileptiform discharges in nerve cells, consequent to post-reperfusion injury, and its pronounced effects on cell viability. Moreover, the analysis revealed that reoxygenation curves for durations of 8, 12, and 24 hours exhibit convergence when OGD interventions are extended to 5 and 6 hours. This pattern suggests that the extended durations of OGD exert a pronounced inhibitory effect on cellular functions, thereby diminishing the relative influence of reoxygenation duration on cell viability.\u003c/p\u003e \u003cp\u003eTo optimally configure the OGD\u0026amp;ED model, it is crucial to pinpoint conditions that minimize cell viability while still allowing for significant recovery. This involves subjecting cells to OGD conditions devoid of glucose and oxygen over six distinct intervals. Following each interval, the culture medium was replaced with a standard solution lacking Mg\u003csup\u003e2+\u003c/sup\u003e for 0-6h, succeeded by reoxygenation phases lasting 8, 12, and 24 hours, respectively.\u003c/p\u003e \u003cp\u003eOur experimental model also incorporated a control group comprising cells cultured in standard medium replete with glucose and oxygen. The influence of varying OGD durations, Mg\u003csup\u003e2+\u003c/sup\u003e-free conditions, and reoxygenation times on cell viability is systematically presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, with a notable reduction in cell viability observed particularly during the shortest reoxygenation period of 8 hours compared to the longer spans of 12 and 24 hours. Statistical analyses confirmed significant disparities across different Mg\u003csup\u003e2+\u003c/sup\u003e-free intervention intervals and between groups subjected to varied durations of Mg\u003csup\u003e2+\u003c/sup\u003e deprivation.\u003c/p\u003e \u003cp\u003eCollectively, the data from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA pinpoint the specific conditions of 3 hours of OGD followed by 8 hours of reoxygenation, in conjunction with 3 hours in a Mg\u003csup\u003e2+\u003c/sup\u003e-free medium, as markedly reducing cell viability. This finding, supported by additional data, underscores a significant decrease in viability immediately following Mg\u003csup\u003e2+\u003c/sup\u003e removal, with a continued decline observed across all subsequent periods. The delineated trend, with OGD duration as a variable, suggests an initial rise followed by a decline in cell viability, with the most significant effects observed at 3 hours of OGD and the most considerable impact of reoxygenation time at 8 hours, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Accordingly, this 3-hour OGD period followed by 8 hours of reoxygenation has been chosen for further investigation into the ramifications of Mg\u003csup\u003e2+\u003c/sup\u003e-free conditions on intracellular Ca\u003csup\u003e2+\u003c/sup\u003e dynamics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration and related protein expression in OGD\u0026amp;ED model\u003c/h2\u003e \u003cp\u003eUpon interruption of cerebral blood flow, mitochondria transition from anaerobic to aerobic metabolism, resulting in an increase in lactic acid production that leads to metabolic acidosis\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn25\" id=\"#FNLinkFn25\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. This shift, coupled with a diminished energy reserve, prompts depolarization of the membrane potential and impairs the functionality of both the sodium-potassium pump (Na+-K+-ATPase) and the calcium pump (Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase) at the cell surface\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn26\" id=\"#FNLinkFn26\"\u003e\u003c/a\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn27\" id=\"#FNLinkFn27\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Consequently, Na+/H\u0026thinsp;+\u0026thinsp;exchangers eject H\u0026thinsp;+\u0026thinsp;and admit an influx of Na+, facilitating the entry of Ca\u003csup\u003e2+\u003c/sup\u003e through the plasma membrane via Na+/Ca\u003csup\u003e2+\u003c/sup\u003e exchangers. Moreover, the inactivation of ATPase contributes to a decrease in Ca\u003csup\u003e2+\u003c/sup\u003e efflux and a restriction in Ca\u003csup\u003e2+\u003c/sup\u003e uptake by the endoplasmic reticulum, culminating in Ca\u003csup\u003e2+\u003c/sup\u003e overload within the cells and impacting the function of mitochondria\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn28\" id=\"#FNLinkFn28\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e is recognized as a pivotal factor in the initiation and progression of epilepsy, given its role in Ca\u003csup\u003e2+\u003c/sup\u003e-dependent signaling pathways that modulate neuronal activity in various ways, ultimately leading to seizures through the enhancement of either focal or generalized neuronal hyperexcitability\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn29\" id=\"#FNLinkFn29\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn30\" id=\"#FNLinkFn30\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Emerging evidence underscores the critical function of Ca\u003csup\u003e2+\u003c/sup\u003e signaling in neurons in relation to both seizures and epilepsy. The orchestration of extraneuronal seizure synchronization involves numerous Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathways, and the interplay between mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e signaling and ROS may precipitate neuronal cell death and epileptic discharges\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn31\" id=\"#FNLinkFn31\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEmploying a Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence probe, intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentrations were quantified from 0 to 6 hours without Mg\u003csup\u003e2+\u003c/sup\u003e during a regimen of 3 hours of OGD followed by 8 hours of reoxygenation, juxtaposing these measurements against a control group. Findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D) revealed a notable increase in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e at 3 hours culturing medium without Mg\u003csup\u003e2+\u003c/sup\u003e, with further elevation at 4 hours. These observations underscore the pronounced impact of OGD and subsequent reoxygenation, particularly during the Mg\u003csup\u003e2+\u003c/sup\u003e-depleted phase, on mitochondrial function and apoptosis, thereby significantly affecting cellular integrity. The apex of Ca\u003csup\u003e2+\u003c/sup\u003e levels at 3 hours post-OGD without Mg\u003csup\u003e2+\u003c/sup\u003e highlights the intervention's efficacy in instigating apoptosis via mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload.\u003c/p\u003e \u003cp\u003eFurther analysis demonstrated that Ca\u003csup\u003e2+\u003c/sup\u003e concentrations in the OGD\u0026amp;ED model significantly exceeded those in the control group, as well as in the groups subjected only to ED and OGD. This suggests that the OGD\u0026amp;ED model precipitates a greater influx of Ca\u003csup\u003e2+\u003c/sup\u003e, prompting a deeper examination of the relationship between Ca\u003csup\u003e2+\u003c/sup\u003e overload and apoptosis. This correlation emphasizes the necessity of understanding mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e dynamics as a potential mechanism underlying cell death and epileptiform activity in the context of cerebral ischemia and reperfusion. Fu et al.\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn32\" id=\"#FNLinkFn32\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e found that the frequency of the CC genotype in PISE patients was significantly higher than that in the non - epileptic control group, and the distribution of the rs2274924C allele was wider in PSE patients. The C allele of rs2274924 is associated with a lower level of magnesium ions in the serum, which increases the intracellular sodium ions, promotes sodium/calcium exchange, increases the concentration of intracellular calcium ions, enhances neuronal excitability, and induces epileptic seizures. It is thus inferred that the polymorphism of TRPM6 rs2274924 and the lower level of serum magnesium ions are potential predictors of post - stroke epilepsy.\u003c/p\u003e \u003cp\u003eThe expression of CaSR was higher than that in the ED and OGD groups, and the Ca\u003csup\u003e2+\u003c/sup\u003e concentration further increased or decreased after the application of CaSR agonists and inhibitors. CaSR is highly expressed in the nervous system and is involved in maintaining intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn33\" id=\"#FNLinkFn33\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn34\" id=\"#FNLinkFn34\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. It is possible that CaSR mediates more Ca\u003csup\u003e2+\u003c/sup\u003e influx. Ca\u003csup\u003e2+\u003c/sup\u003e is a ligand of CaSR, which can act as the first messenger to activate the expression of CaSR in nerve cells, and then induce Ca\u003csup\u003e2+\u003c/sup\u003e overload, resulting in neuronal apoptosis after OGD\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn35\" id=\"#FNLinkFn35\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs a key factor in the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e signaling to maintain intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis, CaMKⅡ can be activated when the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration is induced by neuronal activity\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn36\" id=\"#FNLinkFn36\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e \u003ca class=\"FNLink\" href=\"#Fn37\" id=\"#FNLinkFn37\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e, and then induce neuronal Ca\u003csup\u003e2+\u003c/sup\u003e overload and induce neuronal apoptosis. However, our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed that the expression of CaMKⅡ protein and mRNA was higher than that of ED group, but lower than that of the OGD group. In theory, increased CaMKII activity may underlie seizures, but multiple studies have shown a direct link between reduced CaMKII activity and epilepsy. It has been shown that reduced CaMKII activity and expression represents a homeostasis mechanism that reduces the effects of Ca\u003csup\u003e2+\u003c/sup\u003e overload caused by excess neuronal activity during seizures\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn38\" id=\"#FNLinkFn38\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Therefore, we speculated that the occurrence of ED in the OGD\u0026amp;ED model led to the decrease of CaMKⅡ expression after OGD modeling, but there is no doubt that this is in conflict with the traditional theory that the increase of CaMKII activity may be the basis of epileptic seizures. However, the increase of CaMKII after OGD/R is unquestioned, and CaMKII activation is considered to be a key factor in ischemia-induced neuronal cell death. In ischemic stroke, there is compelling evidence to support rapid activation of CAMKII immediately after hypoxia/reperfusion injury. Activated CaMKII plays a key role in mediating excitotoxic-induced neuronal death\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn39\" id=\"#FNLinkFn39\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Cerebral ischemia-induced translocation of CaMKII into the synaptic membrane may increase neuronal firing rate and Ca\u003csup\u003e2+\u003c/sup\u003e inflow. In addition, current studies have shown that the inactivation of CaMKⅡ can inhibit calcium overload and thus play a protective role in cells. In addition, CaSR and CaMKⅡ interact with each other\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn40\" id=\"#FNLinkFn40\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Studies have found that CaSR activation can up-regulate the expression activity of CaMKⅡ, activate the NLRP3 inflammasome, and aggravate brain injury, while the application of NPS-2143 and KN-93 can inhibit the expression of CaSR and CaMKⅡ, and alleviate OGD injury\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn41\" id=\"#FNLinkFn41\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e \u003ca class=\"FNLink\" href=\"#Fn42\" id=\"#FNLinkFn42\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Cell status and mitochondria damage\u003c/h2\u003e \u003cp\u003eIn the normal group, cells displayed smooth, full surfaces and matured into tight neural networks. In contrast, cells in both the ED and OGD groups exhibited disintegration, collapse, with thin protrusions, atrophy, fractures, necrosis, and reduced adhesion, leading to detachment. This degradation was more pronounced in the OGD\u0026amp;ED group, where cell shedding increased. The use of R568 during reoxygenation, which increased intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and protein expression, aggravated the damage. However, treatments with NPS-2143 and KN-93 improved cell morphology, adhesion, and growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Calcium acts as a critical second messenger in various cellular processes, including neuronal growth, differentiation, and synaptic plasticity. However, when calcium homeostasis is disrupted (for instance, during pathological states leading to calcium overload), it can inhibit neurite outgrowth, alter axonal transport, and impair synaptic formation, ultimately affecting neuronal cell survival and growth. Ureshino\u0026rsquo;s study notes that disruptions in Ca\u003csup\u003e2+\u003c/sup\u003e signaling, often due to overload, can lead to enhanced vulnerability to degeneration by activating destructive enzymes and impairing cellular functions. This disruption is particularly detrimental in neurons, where precise Ca\u003csup\u003e2+\u003c/sup\u003e regulation is crucial for normal function and survival\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn43\" id=\"#FNLinkFn43\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn44\" id=\"#FNLinkFn44\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondrial observations echoed these findings; normal mitochondria were noted in the control group, whereas the ED and OGD groups showed mitochondrial deformation, irregular shapes, and cristae damage. The OGD\u0026amp;ED group faced severe mitochondrial damage, including mitochondriolysis and balloon-like transformations. R568 treatment intensified these effects by raising intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels, while NPS-2143 and KN-93 treatments alleviated the damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Similarly, ROS levels in the OGD\u0026amp;ED group were significantly higher than in the control, ED, and OGD groups, with R568 further elevating ROS levels. Conversely, NPS-2143 and KN-93 treatments effectively reduced ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Excessive calcium uptake by mitochondria, particularly in neurons, can disrupt normal cell function by affecting ATP production, synaptic transmission, and neuronal health. In cases of sustained calcium overload, it can lead to synaptic dysfunction and promote neurodegenerative processes by triggering mitochondrial stress and cell death pathways\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn45\" id=\"#FNLinkFn45\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn46\" id=\"#FNLinkFn46\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study revealed a positive correlation between intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels and mitochondrial damage. In the OGD and ED groups, mitochondrial damage was exacerbated by substantial Ca\u003csup\u003e2+\u003c/sup\u003e influx, further intensified by the activation of the CaSR, which led to even greater Ca\u003csup\u003e2+\u003c/sup\u003e inflow. Mitochondria, critical for energy conversion, ATP production, and the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis and redox states\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn47\" id=\"#FNLinkFn47\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e, are abundant in brain tissue due to its high energy demands, making them essential for the normal function of nerve cells. The extent of mitochondrial damage during brain ischemia-reperfusion injury is a key determinant of brain injury severity. During cerebral ischemia-reperfusion, excessive Ca\u003csup\u003e2+\u003c/sup\u003e accumulates in nerve cells, and mitochondria respond by sequestering this surplus Ca\u003csup\u003e2+\u003c/sup\u003e from the cytoplasm, leading to mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e overload\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn48\" id=\"#FNLinkFn48\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn49\" id=\"#FNLinkFn49\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. This overload is closely linked to epilepsy; recurrent seizures can cause mitochondrial damage, which disrupts Ca\u003csup\u003e2+\u003c/sup\u003e regulation, increases nerve excitability, and perpetuates a vicious cycle of seizures\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn50\" id=\"#FNLinkFn50\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn51\" id=\"#FNLinkFn51\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, mitochondria are principal sources of ROS, which play roles in normal physiological functions. However, our findings indicated more severe mitochondrial damage and increased ROS production in the OGD\u0026amp;ED group. The use of CaSR agonists was associated with higher ROS levels, while CaSR and CaMKⅡ inhibitors reduced intracellular ROS. This increase in ROS during brain injury, particularly at the reperfusion stage, is due to the overactivation of enzymes and pumps hampered by ATP deficiency from ischemia and an inadequate cellular antioxidant capacity, leading to excessive ROS accumulation and mitochondrial dysfunction\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn52\" id=\"#FNLinkFn52\"\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn53\" id=\"#FNLinkFn53\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. The resulting oxidative stress cascade damages mitochondrial and nuclear DNA, promotes the opening of the mitochondrial permeability transition pore, releases apoptosis-inducing factors, and triggers cell death. Additionally, cell and organelle membranes, rich in polyunsaturated fatty acids, are susceptible to ROS-induced damage and lipid peroxidation, increasing membrane permeability and Ca\u003csup\u003e2+\u003c/sup\u003e influx, which further leads to mitochondrial expansion and vacuolation\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn54\" id=\"#FNLinkFn54\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Apoptosis and expression of related proteins\u003c/h2\u003e \u003cp\u003eCompared to the normal group, the apoptosis rate of HT22 cells in the OGD\u0026amp;ED group significantly increased, surpassing rates observed in both the ED and OGD groups. Following R568 treatment in the OGD\u0026amp;ED group, the apoptosis rate further escalated, whereas the application of NPS-2143 and KN-93 ameliorated the apoptosis rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results align with the observed trends in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and its associated protein expression, increased mitochondrial damage, and heightened ROS production, indicating that Ca\u003csup\u003e2+\u003c/sup\u003e may play a role in apoptosis via the mitochondrial pathway in OGD\u0026amp;ED models.\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed that the expression trends of Bax/Bcl2 and Caspase 3 were consistent with the apoptosis rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, after R568 application in the OGD\u0026amp;ED context, Bax/Bcl2 expression did not significantly increase, while Caspase 3 expression further decreased. Immunofluorescence findings indicated that the expression trend of Bcl2 (an anti-apoptotic protein) was inversely related to the apoptosis rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), whereas Caspase 3 expression mirrored the apoptosis rate trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe pro-apoptotic effect of Ca\u003csup\u003e2+\u003c/sup\u003e overload through mitochondrial pathway.\u003c/p\u003e \u003cp\u003eThe key to cell apoptosis is the formation of DNA fragments, and apoptosis involves the activation of DNA endonuclease in cells, leading to DNA fragmentation\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn55\" id=\"#FNLinkFn55\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. In this study, TUNEL was used to detect the apoptosis of each group, based on the detection of dUTP-labeled 3'-OH termini by terminal deoxynucleotidyl transferase. The results of this study showed that Ca\u003csup\u003e2+\u003c/sup\u003e-overload could significantly increase the apoptosis rate of cells. Both Western blot and immunofluorescence results indicated that Bax and Bcl2 proteins were more expressed in the group with higher Ca\u003csup\u003e2+\u003c/sup\u003e and ROS content and more severe mitochondrial damage (the OGD\u0026amp;ED group). ROS accumulation and Ca\u003csup\u003e2+\u003c/sup\u003e-overload in mitochondria can lead to the development of permeability transformation pores and increase mitochondrial permeability, facilitating the entry of pro-apoptotic Bcl2 family proteins Bax and Bak into mitochondria, and the release of cytochrome C into the cytoplasm to activate apoptosis in the mitochondrial pathway\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn56\" id=\"#FNLinkFn56\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Bcl2 can inhibit neuronal apoptosis in cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of mitochondrial membrane permeability, direct antioxidant effects, and inhibition of intracellular calcium overload\u003csup\u003e[\u003c/sup\u003e\u003ca class=\"FNLink\" href=\"#Fn57\" id=\"#FNLinkFn57\"\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. Our results also showed that the expression of Caspase 3 increased in the OGD\u0026amp;ED group and decreased after the application of inhibitors, as Ca\u003csup\u003e2+\u003c/sup\u003e can also mediate endogenous apoptosis through the activation of Caspase 3. In cerebral ischemia-reperfusion injury, the expression ratio of anti-apoptotic protein Bcl2 to pro-apoptotic protein Bax determines whether cells undergo apoptosis. A decrease in the Bcl2/Bax ratio correlates with an increasing trend of apoptosis. Upregulation of Bcl2 and inhibition of Bax and Caspase 3 can inhibit apoptosis of nerve cells. The mechanism pathway diagram is is as follows:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, the results of this study indicate that the optimal intervention time for the OGD\u0026amp;ED model is 3 hours of OGD, 3 hours Mg\u003csup\u003e2+\u003c/sup\u003e-free, and 8 hours of reoxygenation. What\u0026rsquo;s more, our study determined that the combination of OGD and ED causes Ca\u003csup\u003e2+\u003c/sup\u003e overload and increased expression of Ca\u003csup\u003e2+\u003c/sup\u003e related proteins, and the results suggest that Ca\u003csup\u003e2+\u003c/sup\u003e overload is involved in regulating cell death in the mitochondrial pathway in the OGD\u0026amp;ED model, which will lay the foundation for research under OGD\u0026amp;ED conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"589\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull spelling\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eCaMKⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eCalmodulin dependent protein kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eCaSR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eCalcium sensing receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003e4\u0026rsquo;,6-Diamidino-2-phenylindole dihydrochloride\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eDMEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eDulbecco\u0026apos;s Modified Eagle Medium\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eEpileptic discharge\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eReactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eOGD\u0026amp;ED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eOxygen-glucose deprivation combined with epileptiform discharge\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eOGD/R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eOxygen-glucose deprivation / Reoxygenation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003ePISE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003ePost-ischemic stroke epilepsy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eqRT-PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eReal-time Quantitative PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.6367%;\"\u003e\n \u003cp\u003eWB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.3633%;\"\u003e\n \u003cp\u003eWestern blotting\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent to Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in the published papers and the link \u0026nbsp;https://data.mendeley.com/datasets/5699hyhccj/1..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Zhou for critical review of the manuscript and Prof. Qi for assistance with flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChao Gong, Jin Guo, and Shaobo Zhou contributed to the study conception and design. Material preparation, experiments, and data collection and analysis were performed by Chao Gong, Beibei Lian, Pei Zeng, Jiahao Liu, Jiawei Li, Yuanyuan Liu, and Liya Fang. The first draft of the manuscript was written by Chao Gong, Shaobo Zhou and Jin Guo, all authors commented on previous versions of the manuscript. Xunzhong Qi, Luchuan Wang and Jin Guo provided financial support. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Natural Science Foundation of China (No. 81300122), Fund Basic Scientific Research Operating Expenses of Provincial Institutions of Higher Learning in Heilongjiang Province (No. 2021-KYYWF-0609, No. 2022-KYYWF-0653, and 2024-KYYWF-0611) supported this study, Jiamusi University East Pole Academic Team\u0026apos; Children\u0026apos;s Intelligent Rehabilitation Team (No. DJXSTD202413) and Natural Science Foundation of Heilongjiang (No.PL2024H014).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThe Definition and Classification of Cerebral Palsy. Dev Med Child Neurol. 2007 Feb;49(s109):1-44. doi: 10.1111/j.1469-8749.2007.00001.x. PMID: 17371509.\u003c/li\u003e\n\u003cli\u003eMcIntyre S, Goldsmith S, Webb A, Ehlinger V, Hollung SJ, McConnell K, Arnaud C, Smithers-Sheedy H, Oskoui M, Khandaker G, Himmelmann K; Global CP Prevalence Group*. Global prevalence of cerebral palsy: A systematic analysis. Dev Med Child Neurol, 2022, 64(12): 1494-1506.\u003c/li\u003e\n\u003cli\u003eGong C, Liu A, Lian B, Wu X, Zeng P, Hao C, Wang B, Jiang Z, Pang W, Guo J, Zhou S. Prevalence and related factors of epilepsy in children and adolescents with cerebral palsy: a systematic review and meta-analysis. Front Pediatr, 2023, 11: 1189648.\u003c/li\u003e\n\u003cli\u003eZelnik N, Konopnicki M, Bennett-Back O, Castel-Deutsch T, Tirosh E. Risk factors for epilepsy in children with cerebral palsy. Eur J Paediatr Neurol. 2010 Jan;14(1):67-72.\u003c/li\u003e\n\u003cli\u003eGong C, Liu XP, Fang LY, Liu A, Lian BB, Qi XZ, et al. Prevalence of cerebral palsy comorbidities in China: a systematic review and meta-analysis. Front neurol, 2023, 14:1233700.\u003c/li\u003e\n\u003cli\u003e Holtkamp M，Beghi E，Benninger F，et al． European stroke organisation guidelines for the management of post - stroke seizures and epilepsy． Eur Stroke J，2017，2(2): 103-115\u003c/li\u003e\n\u003cli\u003e Oumerzouk J, Vascular Epilepsy. Adv Neurol Neurosci，2021，4(2) : 5-11\u003c/li\u003e\n\u003cli\u003eowska K, Tymianski M. Calcium, ischemia and excitotoxicity. Cell Calcium, 2010, 47(2): 122-129. doi: 10.1016/j.ceca.2010.01.003.\u003c/li\u003e\n\u003cli\u003eg YC. Mitochondrial dysfunction and oxidative stress in seizure-induced neuronal cell death. Acta Neurol Taiwan, 2010, 19(1): 3-15.\u003c/li\u003e\n\u003cli\u003e Walters GC, Usachev YM. Mitochondrial calcium cycling in neuronal function and neurodegeneration. Front Cell Dev Biol, 2023, 11: 1094356.\u003c/li\u003e\n\u003cli\u003e Sarfo FS, Akassi J, Obese V, Adamu S, Agbenorku M, Ovbiagele B. Prevalence and predictors of post-stroke epilepsy among Ghanaian stroke survivors. J Neurol Sci, 2020, 418: 117138.\u003c/li\u003e\n\u003cli\u003e Berg AT, Berkovic SF, Brodie MJ, Buchhalter J, Cross JH, van Emde Boas W, Engel J, French J, Glauser TA, Mathern GW, Mosh\u0026eacute; SL, Nordli D, Plouin P, Scheffer IE. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-2009. Epilepsia, 2010, 51(4): 676-685.\u003c/li\u003e\n\u003cli\u003e Pang W, Lu S, Zheng R, Li X, Yang S, Feng Y, Wang S, Guo J, Zhou S. Investigation into Antiepileptic Effect of Ganoderic Acid A and Its Mechanism in Seizure Rats Induced by Pentylenetetrazole. Biomed Res Int, 2022, 2022: 5940372.\u003c/li\u003e\n\u003cli\u003e Ryou MG, Mallet RT. An In Vitro Oxygen-Glucose Deprivation Model for Studying Ischemia-Reperfusion Injury of Neuronal Cells. Methods Mol Biol. 2018;1717:229-235. doi: 10.1007/978-1-4939-7526-6_18. PMID: 29468596.\u003c/li\u003e\n\u003cli\u003e Tagin M, Shah PS, Lee KS. Magnesium for newborns with hypoxic-ischemic encephalopathy: a systematic review and meta-analysis. Journal of Perinatology, 2013, 33(9): 663-669.\u003c/li\u003e\n\u003cli\u003e Fiest KM, Sauro KM, Wiebe S, Patten SB, Kwon CS, Dykeman J, Pringsheim T, Lorenzetti DL, Jett\u0026eacute; N. Prevalence and incidence of epilepsy: A systematic review and meta-analysis of international studies. Neurology, 2017, 88(3): 296-303.\u003c/li\u003e\n\u003cli\u003e Coultrap SJ, Vest RS, Ashpole NM, Hudmon A, Bayer KU. CaMKII in cerebral ischemia. Acta Pharmacol Sin, 2011, 32(7): 861-8\u003c/li\u003e\n\u003cli\u003e Toussaint F, Charbel C, Allen BG, Ledoux J. Vascular CaMKII: heart and brain in your arteries. Am J Physiol Cell Physiol. 2016, 311(3): C462\u0026ndash;C78.\u003c/li\u003e\n\u003cli\u003e De Baaij JHF, Hoenderop JGJ, Bindels RJM. Magnesium in Man: Implications for Health and Disease. Physiological Reviews, 2017, 95(1): 41-46.\u003c/li\u003e\n\u003cli\u003e Thompson MD, Percy ME, Cole DEC, Bichet DG, Hauser AS, Gorvin CM. G protein-coupled receptor (GPCR) gene variants and human genetic disease. Crit Rev Clin Lab Sci. 2024 Aug;61(5):317-346. doi: 10.1080/10408363.2023.2286606. Epub 2024 Mar 18. PMID: 38497103.\u003c/li\u003e\n\u003cli\u003e Wang J, Xu X, Jia W, Zhao D, Boczek T, Gao Q, Wang Q, Fu Y, He M, Shi R, Tong X, Li M, Tong Y, Min D, Wang W, Guo F. Calcium-/Calmodulin-Dependent Protein Kinase II (CaMKII) Inhibition Induces Learning and Memory Impairment and Apoptosis. Oxid Med Cell Longev. 2021 Dec 23;2021:4635054. doi: 10.1155/2021/4635054. PMID: 34976299; PMCID: PMC8718318.\u003c/li\u003e\n\u003cli\u003e Dong Z, Peng Q, Pan K, Lin W, Wang Y. Microglial and Neuronal Cell Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation Aggravates Cell Injury via Activation of the Caspase-1/GSDMD Signaling Pathway. Neurochem Res, 2023, 48(9): 2660-2673.\u003c/li\u003e\n\u003cli\u003e Qu Y, Liu Y, Zhang H. ALDH2 activation attenuates oxygen-glucose deprivation /reoxygenation-induced cell apoptosis, pyroptosis, ferroptosis and autophagy. Clin Transl Oncol, 2023, 25(11): 3203-3216.\u003c/li\u003e\n\u003cli\u003e Li YH, Zhang S, Tang L, Feng J, Jia J, Chen Y, Liu L, Zhou J. The Role of LincRNA-EPS/Sirt1/Autophagy Pathway in the Neuroprotection Process by Hydrogen against OGD/R-Induced Hippocampal HT22 Cells Injury. J Pers Med, 2023, 13(4): 631.\u003c/li\u003e\n\u003cli\u003e Wang J, Xu J, Dong Y, Su Z, Su H, Cheng Q, Liu X. ADP-ribose transferase PARP16 mediated-unfolded protein response contributes to neuronal cell damage in cerebral ischemia/reperfusion. FASEB J, 2023, 37(2): e22788.\u003c/li\u003e\n\u003cli\u003e Zhou S, Wang SQ, Sun CY, Mao HY, Di WH, Ma XR, Liu L, Liu JX, Wang FF, Kelly P, Sreenivasaprasad P. Investigation into anti-epileptic effect and mechanisms of Ganoderma lucidum polysaccharides in in vivo and in vitro models. Proceedings of the Nutrition Society, 2015, 74(OCE1): E65.\u003c/li\u003e\n\u003cli\u003e Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol, 2014, 115: 157-188.\u003c/li\u003e\n\u003cli\u003e Gorini A, Villa RF. Effect of in vivo treatment of clonidine on ATP-ase's enzyme systems of synaptic plasma membranes from rat cerebral cortex. Neurochem Res, 2001, 26(7): 821-827.\u003c/li\u003e\n\u003cli\u003e Lauritzen M, Mathiesen C, Schaefer K, Thomsen KJ. Neuronal inhibition and excitation, and the dichotomic control of brain hemodynamic and oxygen responses. Neuroimage, 2012, 62(2): 1040-1050.\u003c/li\u003e\n\u003cli\u003e Hu HJ, Song M. Disrupted Ionic Homeostasis in Ischemic Stroke and New Therapeutic Targets. J Stroke Cerebrovasc Dis, 2017, 26(12): 2706-2719.\u003c/li\u003e\n\u003cli\u003e Radak D, Katsiki N, Resanovic I, Jovanovic A, Sudar-Milovanovic E, Zafirovic S, Mousad SA, Isenovic ER. Apoptosis and Acute Brain Ischemia in Ischemic Stroke. Curr Vasc Pharmacol, 2017, 15(2): 115-122.\u003c/li\u003e\n\u003cli\u003e Shutov LP, Kim MS, Houlihan PR, Medvedeva YV, Usachev YM. Mitochondria and plasma membrane Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase control presynaptic Ca\u003csup\u003e2+\u003c/sup\u003e clearance in capsaicin-sensitive rat sensory neurons. J Physiol. 2013, 591(10): 2443-2462.\u003c/li\u003e\n\u003cli\u003e Takemiya T, Yamagata K. Intercellular signaling pathway among Endothelia, astrocytes and neurons in excitatory neuronal damage. Int J Mol Sci, 2013, 14(4):8345-8357.\u003c/li\u003e\n\u003cli\u003e Pei Z, Lee KC, Khan A, Erisnor G, Wang HY. Pathway analysis of glutamate-mediated, calcium-related signaling in glioma progression. Biochem Pharmacol, 2020, 176: 113814.\u003c/li\u003e\n\u003cli\u003e Valero T. Mitochondrial biogenesis: pharmacological approaches. Curr Pharm Des, 2014, 20(35): 5507-5509.\u003c/li\u003e\n\u003cli\u003e FU C Y, CHEN S J, CAI N H, et al. Increased risk of post-stroke epilepsy in Chinese patients with a TRPM6 polymorphism[J]. Neurol Res, 2019, 41(4): 378-383.\u003c/li\u003e\n\u003cli\u003e Wang SQ, Li XJ, Qiu HB, Jiang ZM, Simon M, Ma XR, Liu L, Liu JX, Wang FF, Liang YF, Wu JM, Di WH, Zhou S. Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII \u0026alpha; in epileptic hippocampal neurons. PLoS One, 2014, 9(7): e102161\u003c/li\u003e\n\u003cli\u003e Zhang L, Cao S, Deng S, Yao G, Yu T. Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor. PeerJ, 2016, 4: e2612.\u003c/li\u003e\n\u003cli\u003e Zhen Y, Ding C, Sun J, Wang Y, Li S, Dong L. Activation of the calcium-sensing receptor promotes apoptosis by modulating the JNK/p38 MAPK pathway in focal cerebral ischemia-reperfusion in mice. Am J Transl Res, 2016, 8(2): 911-921.\u003c/li\u003e\n\u003cli\u003e Lu FH, Tian Z, Zhang WH, Zhao YJ, Li HL, Ren H, Zheng HS, Liu C, Hu GX, Tian Y, Yang BF, Wang R, Xu CQ. Calcium-sensing receptors regulate cardiomyocyte Ca\u003csup\u003e2+\u003c/sup\u003e signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation. J Biomed Sci, 2010, 17(1): 50.\u003c/li\u003e\n\u003cli\u003e Takemoto-Kimura S, Suzuki K, Horigane SI, Kamijo S, Inoue M, Sakamoto M, Fujii H, Bito H. Calmodulin kinases: essential regulators in health and disease. J Neurochem, 2017, 141(6): 808-818.\u003c/li\u003e\n\u003cli\u003e Robison AJ. Emerging role of CaMKII in neuropsychiatric disease. Trends Neurosci, 2014, 37(11): 653-662.\u003c/li\u003e\n\u003cli\u003e Zhang X, Connelly J, Levitan ES, Sun D, Wang JQ. Calcium/Calmodulin-Dependent Protein Kinase II in Cerebrovascular Diseases. Transl Stroke Res, 2021, 12(4): 513-529.\u003c/li\u003e\n\u003cli\u003e Chen XH, Chen DT, Huang XM, Chen YH, Pan JH, Zheng XC, Zeng WA. Dexmedetomidine Protects Against Chemical Hypoxia-Induced Neurotoxicity in Differentiated PC12 Cells Via Inhibition of NADPH Oxidase 2-Mediated Oxidative Stress. Neurotox Res, 2019, 35(1):139-149.\u003c/li\u003e\n\u003cli\u003e Zhang X, Hong S, Qi S, Liu W, Zhang X, Shi Z, Chen W, Zhao M, Yin X. NLRP3 Inflammasome Is Involved in Calcium-Sensing Receptor-Induced Aortic Remodeling in SHRs. Mediators Inflamm, 2019, 2019: 6847087.\u003c/li\u003e\n\u003cli\u003e Zhou K, Enkhjargal B, Xie Z, Sun C, Wu L, Malaguit J, Chen S, Tang J, Zhang J, Zhang JH. Dihydrolipoic Acid Inhibits Lysosomal Rupture and NLRP3 Through Lysosome-Associated Membrane Protein-1/Calcium/Calmodulin-Dependent Protein Kinase II/TAK1 Pathways After Subarachnoid Hemorrhage in Rat. Stroke, 2018, 49(1): 175-183.\u003c/li\u003e\n\u003cli\u003e Ureshino RP, Erustes AG, Bassani TB, Wachilewski P, Guarache GC, Nascimento AC, Costa AJ, Smaili SS, Pereira GJDS. The Interplay between Ca\u003csup\u003e2+\u003c/sup\u003e Signaling Pathways and Neurodegeneration. Int J Mol Sci, 2019, 20(23): 6004. doi: 10.3390/ijms20236004.\u003c/li\u003e\n\u003cli\u003e Brini M, Cal\u0026igrave; T, Ottolini D, Carafoli E. Neuronal calcium signaling: function and dysfunction. Cell Mol Life Sci, 2014, 71(15): 2787-2814. doi: 10.1007/s00018-013-1550-7.\u003c/li\u003e\n\u003cli\u003e Verma M, Lizama BN, Chu CT. Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration. Transl Neurodegener, 2022, 11(1): 3.\u003c/li\u003e\n\u003cli\u003e Zhao S, Feng H, Jiang D, Yang K, Wang ST, Zhang YX, Wang Y, Liu H, Guo C, Tang TS. ER Ca\u003csup\u003e2+\u003c/sup\u003e overload activates the IRE1\u0026alpha; signaling and promotes cell survival. Cell Biosci, 2023, 13(1): 123.\u003c/li\u003e\n\u003cli\u003e Wang CH, Wei YH. Role of mitochondrial dysfunction and dysregulation of Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis in the pathophysiology of insulin resistance and type 2 diabetes. J Biomed Sci, 2017, 24(1): 70.\u003c/li\u003e\n\u003cli\u003e Wu M, Gu X, Ma Z. Mitochondrial Quality Control in Cerebral Ischemia-Reperfusion Injury. Mol Neurobiol. 2021, 58(10): 5253-5271.\u003c/li\u003e\n\u003cli\u003e Jia J, Jin H, Nan D, Yu W, Huang Y. New insights into targeting mitochondria in ischemic injury. Apoptosis, 2021, 26(3-4): 163-183.\u003c/li\u003e\n\u003cli\u003e Sumadewi KT, Harkitasari S, Tjandra DC. Biomolecular mechanisms of epileptic seizures and epilepsy: a review. Acta Epileptologica 5, 2023, 28.\u003c/li\u003e\n\u003cli\u003e Madireddy S, Madireddy S. Therapeutic Strategies to Ameliorate Neuronal Damage in Epilepsy by Regulating Oxidative Stress, Mitochondrial Dysfunction, and Neuroinflammation. Brain Sci, 13(5): 784.\u003c/li\u003e\n\u003cli\u003e Sanderson TH, Reynolds CA, Kumar R, Przyklenk K, H\u0026uuml;ttemann M. Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol Neurobiol, 2013, 47(1): 9-23.\u003c/li\u003e\n\u003cli\u003e Wu L, Xiong X, Wu X, Ye Y, Jian Z, Zhi Z, Gu L. Targeting Oxidative Stress and Inflammation to Prevent Ischemia-Reperfusion Injury. Front Mol Neurosci, 2020, 13: 28.\u003c/li\u003e\n\u003cli\u003e Endale HT, Tesfaye W, Mengstie TA. ROS induced lipid peroxidation and their role in ferroptosis. Front Cell Dev Biol, 2023, 11: 1226044.\u003c/li\u003e\n\u003cli\u003e Coletti C, Acosta GF, Keslacy S, Coletti D. Exercise-mediated reinnervation of skeletal muscle in elderly people: An update. Eur J Transl Myol, 2022, 32(1): 10416.\u003c/li\u003e\n\u003cli\u003e Yoshida A, Pommier Y, Ueda T. Endonuclease activation and chromosomal DNA fragmentation during apoptosis in leukemia cells. Int J Hematol. 2006 Jul;84(1):31-37.\u003c/li\u003e\n\u003cli\u003e Zhang L, Li D, Yin L, Zhang C, Qu H, Xu J. Neuroglobin protects against cerebral ischemia/reperfusion injury in rats by suppressing mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal apoptosis through synaptotagmin-1. Environ Toxicol, 2023, 38(8): 1891-1904.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Calcium overload, Oxygen-glucose deprivation, Epileptiform discharge, Mitochondria, CaSR, CaMKⅡ, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6659867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6659867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIschemia and hypoxia are frequently associated with epileptic characteristics, and irregular calcium metabolism can exacerbate neuronal damage. To investigate calcium homeostasis and its effect on neuronal injury, hippocampal HT22 cells were subjected to oxygen-glucose deprivation (OGD), followed by reoxygenation and culturing in Mg\u003csup\u003e2+\u003c/sup\u003e-free medium, to mimic cerebral ischemic and hypoxic brain injury combined with epilepsy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn oxygen-glucose deprivation combined with epileptiform discharge (OGD\u0026amp;ED) model was established to simulate the pathological state of cerebral ischemia-hypoxia combined with epilepsy. The model was evaluated based on cell viability and Ca\u0026sup2;⁺ overload, and the optimal induction conditions were determined as 3-hour OGD, 3-hour culture without Mg\u0026sup2;⁺, and 8-hour reoxygenation. A calcium-sensing receptor (CaSR) agonist (R568) and inhibitor (NPS-2143), combined with a Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) inhibitor (KN-93), were used to study the regulatory effect of Ca\u0026sup2;⁺ overload on the mitochondrial apoptotic pathway. The apoptosis rate, mitochondrial damage, intracellular Ca\u0026sup2;⁺ concentration, and changes in the expression of calcium-related proteins were detected.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared with the single OGD group or the ED group, the OGD\u0026amp;ED group showed a significant increase in Ca\u0026sup2;⁺ influx, more severe cell and mitochondrial damage, and a higher apoptosis rate. The CaSR agonist R568 aggravated Ca\u0026sup2;⁺ overload, while the inhibitor NPS-2143 and the CaMKII inhibitor KN-93 effectively inhibited Ca\u0026sup2;⁺ influx. Ca\u0026sup2;⁺ overload activated the mitochondrial apoptotic pathway, resulting in abnormal expression of apoptosis-related proteins and further aggravating neuronal injury.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the OGD\u0026amp;ED model with an intervention time of OGD3h, Mg\u003csup\u003e2+\u003c/sup\u003e-free, and reoxygenation8h, Ca\u003csup\u003e2+\u003c/sup\u003e overload exacerbates the damage to cells and mitochondria and promotes apoptosis through the mitochondrial pathway.\u003c/p\u003e","manuscriptTitle":"Calcium Overload and Apoptosis in Mitochondrial Pathways under Ischemia, Hypoxia, and Epilepsy-Like Conditions in an In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-07 09:03:14","doi":"10.21203/rs.3.rs-6659867/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-21T23:33:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110578622524806160006268169019436269354","date":"2026-05-11T20:54:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277006422885389883830657603456559391516","date":"2026-05-11T15:14:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234028353884474732488321458094490697934","date":"2026-05-11T13:38:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-17T14:09:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320683554585135538155601518958559907261","date":"2025-07-14T07:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-03T08:25:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-29T22:39:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-26T13:40:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-26T13:37:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Neuroscience","date":"2025-05-14T03:21:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2f27e4e-4db2-4b7e-bfc8-60f587de0cc3","owner":[],"postedDate":"July 7th, 2025","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-21T23:33:31+00:00","index":102,"fulltext":""},{"type":"reviewerAgreed","content":"110578622524806160006268169019436269354","date":"2026-05-11T20:54:08+00:00","index":95,"fulltext":""},{"type":"reviewerAgreed","content":"277006422885389883830657603456559391516","date":"2026-05-11T15:14:26+00:00","index":94,"fulltext":""},{"type":"reviewerAgreed","content":"234028353884474732488321458094490697934","date":"2026-05-11T13:38:25+00:00","index":93,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T09:03:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-07 09:03:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6659867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6659867","identity":"rs-6659867","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}