Chronic magnesium deficiency causes reversible mitochondrial permeability transition pore opening and impairs hypoxia tolerance in the rat heart.

Chronic magnesium (Mg) deficiency induces and exacerbates various cardiovascular diseases. We previously investigated the mechanisms underlying decline in cardiac function caused by chronic Mg deficiency and the effectiveness of Mg supplementation on this decline using the Langendorff-perfused isolated mouse heart model. Herein, we used the Langendorff-perfused isolated rat heart model to demonstrate the chronic Mg-deficient rats (Mg-deficient group) had lower the heart rate (HR) and left ventricular pressure (LVDP) than rats with normal Mg levels (normal group). Furthermore, decline in cardiac function due to hypoxia/reoxygenation injury was significantly greater in the Mg-deficient group than in the normal group. Experiments on mitochondrial permeability transition pore (mPTP) using isolated mitochondria revealed that mitochondrial membrane was fragile in the Mg-deficient group, implying that cardiac function decline through hypoxia/reoxygenation injury is associated with mitochondrial function. Mg supplementation for chronic Mg-deficient rats not only improved hypomagnesemia but also almost completely restored cardiac and mitochondrial functions. Therefore, proactive Mg supplementation in pathological conditions induced by Mg deficiency or for those at risk of developing hypomagnesemia may suppress the development and exacerbation of certain disease states.

Hypomagnesemia and survival in patients with head and neck cancers who received primary concurrent chemoradiation.

BACKGROUND: Prior research has confirmed that persistent hypomagnesemia was predictive of shorter survival among patients with ovarian cancer who received carboplatin-based chemotherapy. In the current retrospective study, the authors examined the association between hypomagnesemia and survival in patients with head and neck cancer who received concurrent chemoradiation with weekly infusions of cisplatin and/or carboplatin. METHODS: Patients with head and neck cancers who had undergone chemoradiation with cisplatin and/or carboplatin between January 1, 2010, and December 31, 2014, were included. Patients were aged ≥18 years with pathology of squamous cell carcinoma of the larynx, oral cavity, or oropharynx who had received at least 30 fractions of radiotherapy with concurrent weekly cisplatin and/or carboplatin. Pathology features, laboratory results, Eastern Cooperative Oncology Group performance status, social histories, and survival were recorded. The association between hypomagnesemia and survival was analyzed controlling for known prognostic factors. RESULTS: The final cohort consisted of 439 patients with a median age of 59 years. A greater frequency of hypomagnesemia during the treatment course was found to be significantly associated with shorter survival (hazard ratio [HR], 1.13; P = .033) independent of age (HR, 1.65; P = .042), cancer site (nonoropharynx vs oropharynx: HR, 2.15 [P = .003]), Eastern Cooperative Oncology Group performance status (>1 vs ≤1: HR, 2.64 [P < .001]), and smoking history (smoker vs nonsmoker: HR, 1.88 [P = .012]). In addition, more severe hypomagnesemia was associated with shorter survival compared with the milder form. CONCLUSIONS: The frequency and severity of hypomagnesemia during treatment are prognostic of survival for patients with head and neck cancers who are receiving concurrent chemoradiation with cisplatin and/or carboplatin. A prospective study is needed to investigate the impact of the prevention of hypomagnesemia on survival in this patient population.

[Pathogenetic aspects of magnesium deficiency in connective tissue dysplasia syndrome].

The problem of connective tissue dysplasia is currently becoming particularly relevant because of significant increase of individuals with characteristic abnormalities in the structure of connective tissue. The lack of some micronutrients, arising during ontogenesis in the organism, can determine a high risk of worsening connective tissue homeostasis. Recently, the decisive role of magnesium deficiency in the progression of this disease has been demonstrated. The aim of the study was to substantiate the need for magnesium diet therapy in individuals with connective tissue dysplasia basing on the study of the pathogenetic significance of magnesium deficiency in this pathology. Material and methods. The electronic resources of the portals PubMed-NCBI, MEDLINE, the Scientific Electronic Library eLIBRARY.RU, CyberLeninka and the Google Academy were used. Results and discussion. The analysis of the obtained data made it possible to identify fundamentally new provisions on the main mechanisms of the magnesium influence on the metabolic state of all components of connective tissue. It was proved that magnesium deficiency is a predictor of worsening connective tissue homeostasis, increasing in the number of dysplastic symptoms and their severity. This pathogenetically justifies prescribing a balanced diet to patients with connective tissue dysplasia, including products rich in magnesium, taking into account its recommended daily intake, depending on age of patients. Conclusion. Adequate daily intake of magnesium will increase the mechanical properties and functionality of the connective tissue, and should be recommended for patients with connective tissue dysplasia to prevent the development of complications, maintain the quality of life and improve the prognosis for this disease.

Effects of Magnesium Deficiency on Mechanisms of Insulin Resistance in Type 2 Diabetes: Focusing on the Processes of Insulin Secretion and Signaling.

Magnesium (Mg2+) is an essential mineral for human health and plays an important role in the regulation of glucose homeostasis and insulin actions. Despite the widespread clinical evidences for the association of Mg2+ deficiency (MgD) and type 2 diabetes mellitus (T2D), molecular mechanisms by which Mg2+ contributes to insulin resistance (IR) are still under discussion. Mg2+ regulates electrical activity and insulin secretion in pancreatic beta-cells. Intracellular Mg2+ concentrations are critical for the phosphorylation of the insulin receptor and other downstream signal kinases of the target cells. Low Mg2+ levels result in a defective tyrosine kinase activity, post-receptor impairment in insulin action, altered cellular glucose transport, and decreased cellular glucose utilization, which promotes peripheral IR in T2D. MgD triggers chronic systemic inflammation that also potentiates IR. People with T2D may end up in a vicious circle in which MgD increases IR and IR causes MgD, that requires periodic monitoring of serum Mg2+ levels.

Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions.

The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet-induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+-induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency-induced skeletal diseases.

Ionized Magnesium and Regional Citrate Anticoagulation for Continuous Renal Replacement Therapy.

BACKGROUND: The regional citrate anticoagulation (RCA) induces changes in total (Catot) and ionized (Ca2+) calcium. As of now, we do not have much information about parallel changes of total (Mgtot) and ionized (Mg2+) magnesium. METHODS: The authors compared changes of Mg2+ and Mgtot with changes of Ca2+ and Catot in 32 critically ill patients on 4% trisodium citrate (4% TSC) with calcium-free fluids. RESULTS: The median continuous venovenous hemodiafiltration balance of Mgtot was -0.91 (-1.18 to -0.53) mmol/h compared to the median balance of Catot 0.86 (0.08-1.55) mmol/h. Postfilter Mg2+ decreased by 68.3% (70.8-65.6) in parallel (r = 0.41, p = 0.03) to decrease of postfilter Ca2+ (by 70.2% (73.0-66.1)) and was significantly related to the postfilter Ca2+ (r = 0.50, p < 0.001). The decrease of prefilter to postfilter Ca2+ correlated to a dosage of 4% TSC per blood flow (r = 0.37, p = 0.04). CONCLUSIONS: The loss of Mgtot during RCA is not covered by magnesium concentration in ordinary dialysis/substitution fluid and may lead to the depletion of total body magnesium. The postfilter Mg2+ is significantly related to the postfilter Ca2+. Video Journal Club "Cappuccino with Claudio Ronco" at http://www.karger.com/?doi = 440972.

[TREATMENT OF A NEWBORN BABIES FOR AN ACUTE DISORDER OF THE BRAIN BLOOD CIRCULATION OF A HEMORRHAGIC TYPE].

A timely and adequate application of complex of conservative and surgical measures determines at large the result of treatment of a newbor babies, suffering perinatal intracranial hematoma. The treatment includes, besides neurosurgical manipulations and operations, providing of evacuation of the blood extrused, the intracranial pressure normalization, liquorocirculation restoration, correction of hemodynamical and metabolic disorders, antiedematous, membrane-stabilizing and anticonvulsant therapy. A control of metabolic disorders, as well as especially hypoglycemia, hypocalcemia, hypomagnesemia, hypopyridoxinemia constitutes a leading moment of the treatment

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.

Severe hypomagnesaemia causing reversible cerebellopathy.

Hypomagnesaemia is common among hospitalised patients and is often under-recognised. Chronic alcohol abuse and alcohol withdrawal are known causes for severe hypomagnesaemia. Hypomagnesaemia can present with cardiac arrhythmias, seizures and other neurological symptoms, among which ataxia. We present a 57-year-old man with a history of chronic alcohol abuse who developed a subacute cerebellar syndrome with hypertension after alcohol withdrawal. A severe hypomagnesaemia of 0.19 mmol/L (normal values 0.70-1.10) was found. MRI showed diffuse, T2 hyperintense lesions in and swelling of the cerebellum. Symptoms, hypertension and MRI abnormalities significantly improved rapidly after intravenous magnesium supplementation. Hypomagnesaemia can cause a subacute, cerebellar syndrome and hypertension. Symptoms, hypertension and MRI abnormalities can be reversed with rapid magnesium supplementation. MRI abnormalities are similar to those caused by vascular endothelial dysregulation seen in posterior reversible encephalopathy syndrome (PRES). A similar case was recently described. We confirm that magnesium is likely to be involved in the pathophysiology of PRES.

Silencing TRPM7 mimics the effects of magnesium deficiency in human microvascular endothelial cells.

Evidence has accumulated to suggest that magnesium might play a role in controlling angiogenesis. Since microvascular endothelial cells are protagonists in this process, we investigated the behavior of these cells cultured in low extracellular magnesium or silenced for its transporter Transient Receptor Potential Melastatin (TRPM)7, essential for cellular magnesium homeostasis. In particular, we focused on some crucial steps of the angiogenic process, i.e. proliferation, migration, protease production and organization in tridimensional structures. Silencing TRPM7 mimics the effects of low extracellular magnesium on human microvascular endothelial cells (HMEC). Indeed, while no effects were observed on the production of metalloproteases and on tridimensional organization on matrigel, both magnesium deficiency and silencing of TRPM7 impair cell migration and inhibit growth by arresting the cells in the G0/G1 and G2/M phases of the cell cycle. Since low extracellular magnesium markedly decreases TRPM7 in HMEC, we suggest that TRPM7 downregulation might mediate low magnesium-induced inhibition of cell growth and migration. Human endothelial cells from the umbilical vein are growth inhibited by low magnesium and growth stimulated after TRPM7 silencing. An impairment of ERK phosphorylation in HMEC silencing TRPM7 is responsible, in part, for the different proliferative behavior of these two cell types. We broadened our studies also to endothelial colony-forming cells and found that they are sensitive to fluctuations of the concentrations of extracellular magnesium, while their proliferation rate is not modulated by TRPM7 silencing. Our results point to magnesium and TRPM7 as a modulators of the angiogenic phenotype of microvascular endothelial cells.

Influences of Hypomagnesemia on Optic Nerve and Retinal Vascular Structure Determined Using Optical Coherence Tomography (OCT) Angiography.

Purpose: To evaluate the effects of low blood magnesium levels on the optic nerve, retina, and retinal vascular structure.Methods: This observational and cross-sectional study was conducted between June 2019 and May 2020 with participants aged 20-39 years, who had a visual acuity of ≥20/20, the axial length of 22-24.5 mm, refractive defect spherical equivalent of ≤±3D, and intraocular pressure of ≤21 mm Hg. All participants had a complaint of twitching in their eyes. The participants with normal serum magnesium levels constituted the control group, and patients with hypomagnesemia constituted the patient group. Updated AngioScan software (Navis ver. 1.8.0.) of Nidek's RS-3000 Advance system was used to analyze the spectral domain-optical coherence tomography (SD-OCT) and optical coherence tomography-angiography (OCT-A) images.Results: A total of 100 right eyes of 100 individuals was included in the study. The mean retinal nerve fiber layer (RNFL) thickness was 108.52 ± 12.46 µm in the control group, and 97.3 ± 9.7 µm in the hypomagnesemia group (P < .001). In the control group, the global superficial capillary plexus (SCP) and deep capillary plexus (DCP) vessel densities (VDs) were 41.92 ± 2.29, and 37.54 ± 3.83, respectively. In the patient group, the global SCP and DCP VDs were 37.66 ± 3.14, and 32.95 ± 5.57, respectively. The SCP and DCP VD percentages were significantly lower in the patient group. The mean foveal avascular zone (FAZ) area, perimeter and circularity index (CI) were 0.32 ± 0.13 mm2, 2.89 ± 0.59 mm, and 0.52 ± 0.09, respectively, for the control group and 0.38 ± 0.11 mm2, 2.99 ± 0.64 mm, and 0.38 ± 0.1, respectively, for the patient group. The FAZ area and perimeter were significantly higher (P = .013 and P = .001) and FAZ CI was significantly lower (P < .001) in the patients with hypomagnesemia.Conclusion: Our study revealed that OCT and OCT-A measurements may be used in the determination of the optic nerve and retinal vascular structure changes in hypomagnesemia.

Cyclin M2 (CNNM2) knockout mice show mild hypomagnesaemia and developmental defects.

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/- mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2-/- pups were born alive. The Cnnm2-/- pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/- mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/- mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/- mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

Novel compound heterozygous mutations of CLDN16 in a patient with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

BACKGROUND: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive tubulopathy characterized by excessive urinary wasting of magnesium and calcium, bilateral nephrocalcinosis, and progressive chronic renal failure in childhood or adolescence. FHHNC is caused by mutations in CLDN16 and CLDN19, which encode the tight-junction proteins claudin-16 and claudin-19, respectively. Most of these mutations are missense mutations and large deletions are rare. METHODS: We examined the clinical and biochemical features of a Spanish boy with early onset of FHHNC symptoms. Exons and flanking intronic segments of CLDN16 and CLDN19 were analyzed by direct sequencing. We developed a new assay based on Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) to investigate large CLDN16 deletions. RESULTS: Genetic analysis revealed two novel compound heterozygous mutations of CLDN16, comprising a missense mutation, c.277G>A; p.(Ala93Thr), in one allele, and a gross deletion that lacked exons 4 and 5,c.(840+25_?)del, in the other allele. The patient inherited these variants from his mother and father, respectively. CONCLUSIONS: Using direct sequencing and our QMPSF assay, we identified the genetic cause of FHHNC in our patient. This QMPSF assay should facilitate the genetic diagnosis of FHHNC. Our study provided additional data on the genotypic spectrum of the CLDN16 gene.

Magnesium: The overlooked electrolyte in blood cancers?

Magnesium is an important element that has essential roles in the regulation of cell growth, division, and differentiation. Mounting evidence in the literature suggests an association between hypomagnesemia and all-cause mortality. In addition, epidemiologic studies have demonstrated that a diet poor in magnesium increases the risk of developing cancer, highlighting its importance in the field of hematology and oncology. In solid malignancies, hypomagnesemia at diagnosis portends a worse prognosis. However, little is known about prognosis in patients with hypomagnesemia and blood cancers in general; lymphoma more specifically. Hypomagnesemia has been associated with a higher viral load of the Epstein Barr virus, a virus associated with a multitude of hematologic malignancies. The role of magnesium in the immune system has been further elucidated in studies of patients with a rare primary immunodeficiency known as XMEN disease (X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus (EBV) infection, and Neoplasia disease). These patients have a mutation in the MAGT1 gene, which codes for a magnesium transporter. The mutation leads to impaired T cell activation and an increased risk of developing hematologic malignancies. In this review we discuss the relevance of magnesium as an electrolyte, current measurement techniques, and the known data related to cause and prognosis of blood cancers. The goal is to use these data to stimulate additional high-quality and well powered studies to further investigate the role of magnesium in preventing cancer and improving outcomes of patients with malignancy and concomitant magnesium deficiency.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Magnesium deficiency and hypertension: correlation between magnesium-deficient diets and microcirculatory changes in situ.

Rats maintained for 12 weeks on diets moderately or more severely deficient in magnesium showed significant elevations in arterial blood pressure compared to control animals. Examination of the mesenteric microcirculation in situ revealed that dietary magnesium deficiency resulted in reduced capillary, postcapillary, and venular blood flow concomitant with reduced terminal arteriolar, precapillary sphincter, and venular lumen sizes. The greater the degree of dietary magnesium deficiency the greater the reductions in microvascular lumen sizes. These findings may provide a rationale for the etiology, as well as treatment, of some forms of hypertensive vascular disease.

Magnesium deficiency affects mammary epithelial cell proliferation: involvement of oxidative stress.

Low Mg availability reversibly inhibited the growth of mammary epithelial HC11 cells by increasing the number of cells in the G0/G1 phase of the cell cycle. Because low Mg has been reported to promote oxidative reactions, we considered that low Mg-dependent growth arrest was mediated by oxidative stress. Surprisingly, both dichlorofluorescein-detectable reactive oxygen species and hydrogen peroxide-induced oxidative DNA damage were found to be lower in cells cultured in low Mg than in cells grown under control or high-Mg conditions. Gene expression profiling of low- and high-Mg cells showed the modulation of several genes, some regulating cell proliferation. In addition, low Mg cells also displayed overexpression of glutathione S-transferase (GST), leading to increased enzymatic activity. Of note, GST has been shown to modulate cell growth; therefore, we suggest that in low-Mg cells, GST upregulation might have a dual role in protecting against oxidative stress and in modulating cell proliferation.

CNNM2 homozygous mutations cause severe refractory hypomagnesemia, epileptic encephalopathy and brain malformations.

Magnesium (Mg2+) plays a crucial role in many biological processes especially in the brain, heart and skeletal muscle. Mg2+ homeostasis is regulated by intestinal absorption and renal reabsorption, involving a combination of different epithelial transport pathways. Mutations in any of these transporters result in hypomagnesemia with variable clinical presentations. Among these, CNNM2 is found along the basolateral membrane of distal tubular segments where it is involved in Mg2+ reabsorption. To date, heterozygous mutations in CNNM2 have been associated with a variable phenotype, ranging from isolated hypomagnesemia to intellectual disability and epilepsy. The only homozygous mutation reported so far, is responsible for hypomagnesemia associated with a severe neurological phenotype characterized by refractory epilepsy, microcephaly, severe global developmental delay and intellectual disability. Here, we report the second homozygous CNNM2 mutation (c.1642G > A,p.Val548Met) in a Moroccan patient, presenting with hypomagnesemia and severe epileptic encephalopathy. Thus, we review and discuss the phenotypic spectrum associated with CNNM2 mutations.

Time course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus: role of NMDA receptor activation and NMDA dependent calcium entry.

The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg2+) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and fluorescein diacetate-propidium iodide staining. Effects of low Mg2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and time-dependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium (Ca2+) that entered primarily through the N-methyl-d-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by co-incubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular (Ca2+) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca2+ channels. Interestingly this NMDA-Ca2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca2+ transduction pathway leading to neuronal death in a time-dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus.