Convulsive Stress Mimics Brain Hypoxia and Promotes the P-Glycoprotein (P-gp) and Erythropoietin Receptor Overexpression. Recombinant Human Erythropoietin Effect on P-gp Activity

Erythropoietin (EPO) is not only a hormone that promotes erythropoiesis but also has a neuroprotective effect on neurons attributed to its known anti-apoptotic action. Previously, our group has demonstrated that recombinant-human EPO (rHu-EPO) can protect neurons and recovery motor activity in a chemical focal brain hypoxia model (Merelli et al., 2011). We and others also have reported that repetitive seizures can mimic a hypoxic- like condition by HIF-1α nuclear translocation and high neuronal expression P-gp. Here, we report that a single 20-min status epilepticus (SE) induces P-gp and EPO-R expression in cortical pyramidal neurons and only P-gp expression in astrocytes. In vitro, excitotoxic stress (300 μM glutamate, 5 min), can also induce the expression of EPO-R and P-gp simultaneously with both HIF-1α and NFkB nuclear translocation in primary cortical neurons. Primary astrocytes exposed to chemical hypoxia with CoCl2 (0.3 mM, 6 h) increased P-gp expression as well as an increased efflux of Rhodamine 123 (Rho123) that is a P-gp substrate. Tariquidar, a specific 3er generation P-gp-blocker was used as an efflux inhibitor control. Astrocytes treated with rHu-EPO showed a significant recovery of the Rho123 retention in a similar way as seen by Tariquidar, demonstrating for first time that rHu-EPO can inhibit the P-gp-dependent efflux activity. Taking together, these data suggest that stimulation of EPO depending signaling system could not only play a central role in brain cell protection, but this system could be a new tool for reverse the pharmacoresistant phenotype in refractory epilepsy as well as in other pharmacoresistant hypoxic brain diseases expressing P-gp.Fil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Ramos, Alberto Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Auzmendi, Jerónimo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Progressive heart P-glycoprotein (P-gp) overexpression after experimental repetitive seizures (ERS) associated with fatal status epilepticus (FSE): Is it related with SUDEP?

Patients with refractory epilepsy (RE) have increased risk of Sudden Unexpected Death in Epilepsy (SUDEP), where acute and fatal heart failure is suspected. High seizure frequency, polypharmacy, changes in dosing, persistent low AED levels or poor adherence to therapy are the greatest risk factors of SUDEP and are also features observed in RE. We evaluated the progressive P-gp overexpression in heart, related with the development of fatal status epilepticus (FSE) after experimental repetitive seizures (ERS). Male Wistar rats (180–230g) were daily injected (i.p) with Pentylenetetrazole (PTZ; 45mg/kg; n=18) or saline (Controls; n=6). Severity of seizures was recorded. Four PTZ-treated rats were sacrificed at 4th-7th day respectively. Ten remaining rats, underwent same treatment until develop fatal status epilepticus (FSE). Brains and hearts were studied by immunofluorecent method for P-glycoptrotein (P-gp) expression. Seizures were observed each day of PTZ treatment, associated with a progressive P-gp overexpression in heart and FSE at 9th day. Using the same PTZ model, we previously demonstrated that progressive brain P-gp overexpression contributes to cell membrane depolarization of hippocampus and neocortex. These are the first evidences showing that ERS induces a simultaneous and progressive P-gp overexpression in brain and heart associated with FSE, and suggests a role for this pattern expression of P-gp as risk factor for death during SE. The simultaneous and spontaneous death of all animals during SE observed only at day 9th, suggest that a higher P-gp overexpression in cardiomyocytes could play a role in SUDEP, however, because it is only a study descriptive, further researches are needed to confirm this hypothesis.Fil: Auzmendi, Jerónimo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Girardi, Elena Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Orozco Suárez, Sandra. Centro Médico Nacional Siglo XXI; MéxicoFil: Rocha Arrieta, Luisa Lilia. Centro de Investigación y de Estudios Avanzados. Departamento de Farmacobiología; MéxicoFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Fundación Investigar; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Cannabidiol (Cbd) alters the functionality of neutrophils (pmn). implications in the refractory epilepsy treatment

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti‐epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1‐CB2) or multiple non‐canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P‐glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration‐dependent manner, while low CBD concentration decreased PMN viability in a time‐dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti‐inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.Fil: Taborda Gómez, Claudia Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lairion, Fabiana Norma. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Repetto, Marisa Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Ettcheto, Miren. Universidad de Barcelona; EspañaFil: Merelli, Amalia Margarita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; ArgentinaFil: Auzmendi, Jerónimo Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Myocardial iron overload in an experimental model of sudden unexpected death in epilepsy

Uncontrolled repetitive generalized tonic-clonic seizures (GTCS) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). GTCS can be observed in models such as Pentylenetetrazole kindling (PTZ-K) or pilocarpine-induced Status Epilepticus (SE-P), which share similar alterations in cardiac function, with a high risk of SUDEP. Terminal cardiac arrhythmia in SUDEP can develop as a result of a high rate of hypoxic stress-induced by convulsions with excessive sympathetic overstimulation that triggers a neurocardiogenic injury, recently defined as ?Epileptic Heart? and characterized by heart rhythm disturbances, such as bradycardia and lengthening of the QT interval. Recently, an iron overload-dependent form of non-apoptotic cell death called ferroptosis was described at the brain level in both the PTZ-K and SE-P experimental models. However, seizure-related cardiac ferroptosis has not yet been reported. Iron overload cardiomyopathy (IOC) results from the accumulation of iron in the myocardium, with high production of reactive oxygen species (ROS), lipid peroxidation, and accumulation of hemosiderin as the final biomarker related to cardiomyocyte ferroptosis. Iron overload cardiomyopathy is the leading cause of death in patients with iron overload secondary to chronic blood transfusion therapy; it is also described in hereditary hemochromatosis. GTCS, through repeated hypoxic stress, can increase ROS production in the heart and cause cardiomyocyte ferroptosis. We hypothesized that iron accumulation in the ?Epileptic Heart? could be associated with a terminal cardiac arrhythmia described in the IOC and the development of state-potentially in the development of SUDEP. Using the aforementioned PTZ-K and SE-P experimental models, after SUDEP-related repetitive GTCS, we observed an increase in the cardiac expression of hypoxic inducible factor 1α, indicating hypoxic-ischemic damage, and both necrotic cells and hemorrhagic areas were related to the possible hemosiderin production in the PTZ-K model. Furthermore, we demonstrated for the first time an accumulation of hemosiderin in the heart in the SE-P model. These results suggest that uncontrolled recurrent seizures, as described in refractory epilepsy, can give rise to high hypoxic stress in the heart, thus inducing hemosiderin accumulation as in IOC, and can act as an underlying hidden mechanism contributing to the development of a terminal cardiac arrhythmia in SUDEP. Because iron accumulation in tissues can be detected by non-invasive imaging methods, cardiac iron overload in refractory epilepsy patients could be treated with chelation therapy to reduce the risk of SUDEP.Fil: Akyuz, Enes. Yozgat Bozok University; TurquíaFil: Doganyigit, Zuleyha. Yozgat Bozok University; TurquíaFil: Eroglu, Ece. Yozgat Bozok University; TurquíaFil: Moscovicz, Franco. Universidad de Buenos Aires; ArgentinaFil: Merelli, Amalia Margarita. Universidad de Buenos Aires; ArgentinaFil: Lazarowski, Alberto Jorge. Universidad de Buenos Aires; ArgentinaFil: Auzmendi, Jerónimo Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Fisiopatología y Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin