Time- and Region-Specific Selection of Reference Genes in the Rat Brain in the Lithium-Pilocarpine Model of Acquired Temporal Lobe Epilepsy.

Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium-pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium-pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.

Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model.

Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

Theoretical Analysis of the Influence of Spacers on Salt Ion Transport in Electromembrane Systems Considering the Main Coupled Effects.

This article considers a theoretical analysis of the influence of the main coupled effects and spacers on the transfer of salt ions in electromembrane systems (EMS) using a 2D mathematical model of the transfer process in a desalting channel with spacers based on boundary value problems for the coupled system of Nernst-Planck-Poisson and Navier-Stokes equations. The basic patterns of salt ion transport have been established, taking into account diffusion, electromigration, forced convection, electroconvection, dissociation/recombination reactions of water molecules, as well as spacers located inside the desalting channel. It has been shown that spacers and taking into account the dissociation/recombination reaction of water molecules significantly change both the formation and development of electroconvection. This article confirms the fact of the exaltation of the limiting current studied by Harkatz, where it is shown that the current (flux) of salt ions increases when the dissociation reaction begins by a certain value called the exaltation current, which is proportional to the flow of water dissociation products. A significant combined effect of electroconvection and dissociation/recombination reactions as well as the spacer system in the desalting channel on the transport of salt ions are shown. The complex, nonlinear, and non-stationary interaction of all the main effects of concentration polarization and spacers in the desalting channel are also considered in the work.

Investigating the Metabolism of Plants Germinated in Heavy Water, D2O, and H218O-Enriched Media Using High-Resolution Mass Spectrometry.

Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, Lepidium sativum, germinated in D2O and H218O-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.

Untargeted Lipidomics after D2O Administration Reveals the Turnover Rate of Individual Lipids in Various Organs of Living Organisms.

The administration of low doses of D2O to living organisms was used for decades for the investigation of metabolic pathways and for the measurement of the turnover rate for specific compounds. Usually, the investigation of the deuterium uptake in lipids is performed by measuring the deuteration level of the palmitic acid residue using GC-MS instruments, and to our knowledge, the application of the modern untargeted LC-MS/MS lipidomics approaches was only reported a few times. Here, we investigated the deuterium uptake for >500 lipids for 13 organs and body liquids of mice (brain, lung, heart, liver, kidney, spleen, plasma, urine, etc.) after 4 days of 100% D2O administration. The maximum deuteration level was observed in the liver, plasma, and lung, while in the brain and heart, the deuteration level was lower. Using MS/MS, we demonstrated the incorporation of deuterium in palmitic and stearic fragments in lipids (PC, PE, TAG, PG, etc.) but not in the corresponding free forms. Our results were analyzed based on the metabolic pathways of lipids.

Beneficial Effects of Probiotic Bifidobacterium longum in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy in Rats.

Epilepsy is a challenging brain disorder that is often difficult to treat with conventional therapies. The gut microbiota has been shown to play an important role in the development of neuropsychiatric disorders, including epilepsy. In this study, the effects of Bifidobacterium longum, a probiotic, on inflammation, neuronal degeneration, and behavior are evaluated in a lithium-pilocarpine model of temporal lobe epilepsy (TLE) induced in young adult rats. B. longum was administered orally at a dose of 109 CFU/rat for 30 days after pilocarpine injection. The results show that B. longum treatment has beneficial effects on the TLE-induced changes in anxiety levels, neuronal death in the amygdala, and body weight recovery. In addition, B. longum increased the expression of anti-inflammatory and neuroprotective genes, such as Il1rn and Pparg. However, the probiotic had little effect on TLE-induced astrogliosis and microgliosis and did not reduce neuronal death in the hippocampus and temporal cortex. The study suggests that B. longum may have a beneficial effect on TLE and may provide valuable insights into the role of gut bacteria in epileptogenesis. In addition, the results show that B. longum may be a promising drug for the comprehensive treatment of epilepsy.

Mathematical Modeling of the Influence of the Karman Vortex Street on Mass Transfer in Electromembrane Systems.

In electromembrane systems, the transfer of ions near ion-exchange membranes causes concentration polarization, which significantly complicates mass transfer. Spacers are used to reduce the effect of concentration polarization and increase mass transfer. In this article, for the first time, a theoretical study is carried out, using a two-dimensional mathematical model, of the effect of spacers on the mass transfer process in the desalination channel formed by anion-exchange and cation-exchange membranes under conditions when they cause a developed Karman vortex street. The main idea is that, when the separation of vortices occurs on both sides in turn from the spacer located in the core of the flow where the concentration is maximum, the developed non-stationary Karman vortex street ensures the flow of the solution from the core of the flow alternately into the depleted diffusion layers near the ion-exchange membranes. This reduces the concentration polarization and, accordingly, increases the transport of salt ions. The mathematical model is a boundary value problem for the coupled system of Nernst-Planck-Poisson and Navier-Stokes equations for the potentiodynamic regime. The comparison of the current-voltage characteristics calculated for the desalination channel with and without a spacer showed a significant increase in the intensity of mass transfer due to the development of the Karman vortex street behind the spacer.

Alterations in the Properties of the Rat Hippocampus Glutamatergic System in the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy.

Status epilepticus (SE) triggers many not yet fully understood pathological changes in the nervous system that can lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in the lithium-pilocarpine model of temporal lobe epilepsy in rats. The studies were performed 1 day (acute phase), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. According to RT-qPCR data, expression of the genes coding for the AMPA receptor subunits GluA1 and GluA2 was downregulated in the latent phase, which may lead to the increased proportion of calcium-permeable AMPA receptors that play an essential role in the pathogenesis of many CNS diseases. The efficiency of excitatory synaptic neurotransmission in acute brain slices was decreased in all phases of the model, as determined by recording field responses in the CA1 region of the hippocampus in response to the stimulation of Schaffer collaterals by electric current of different strengths. However, the frequency of spontaneous excitatory postsynaptic potentials increased in the chronic phase, indicating an increased background activity of the glutamatergic system in epilepsy. This was also evidenced by a decrease in the threshold current causing hindlimb extension in the maximal electroshock seizure threshold test in rats with temporal lobe epilepsy compared to the control animals. The results suggest a series of functional changes in the properties of glutamatergic system associated with the epilepsy development and can be used to develop the antiepileptogenic therapy.

Imbalance of Angiogenic and Growth Factors in Placenta in Maternal Hyperhomocysteinemia.

Numerous studies have shown that various adverse factors of different nature and action mechanisms have similar negative influence on placental angiogenesis, resulting in insufficiency of placental blood supply. One of the risk factors for pregnancy complications with placental etiology is an increased level of homocysteine in the blood of pregnant women. However, the effect of hyperhomocysteinemia (HHcy) on the development of the placenta and, in particular, on the formation of its vascular network is at present poorly understood. The aim of this work was to study the effect of maternal HHcy on the expression of angiogenic and growth factors (VEGF-A, MMP-2, VEGF-B, BDNF, NGF), as well as their receptors (VEGFR-2, TrkB, p75NTR), in the rat placenta. The effects of HHcy were studied in the morphologically and functionally different maternal and fetal parts of the placenta on the 14th and 20th day of pregnancy. The maternal HHcy caused increase in the levels of oxidative stress and apoptosis markers accompanied by an imbalance of the studied angiogenic and growth factors in the maternal and/or fetal part of the placenta. The influence of maternal HHcy in most cases manifested in a decrease in the protein content (VEGF-A), enzymatic activity (MMP-2), gene expression (VEGFB, NGF, TRKB), and accumulation of precursor form (proBDNF) of the investigated factors. In some cases, the effects of HHcy differed depending on the placental part and stage of development. The influence of maternal HHcy on signaling pathways and processes controlled by the studied angiogenic and growth factors could lead to incomplete development of the placental vasculature and decrease in the placental transport, resulting in fetal growth restriction and impaired fetal brain development.

Maternal Hyperhomocysteinemia Disturbs the Mechanisms of Embryonic Brain Development and Its Maturation in Early Postnatal Ontogenesis.

Maternal hyperhomocysteinemia causes the disruption of placental blood flow and can lead to serious disturbances in the formation of the offspring's brain. In the present study, the effects of prenatal hyperhomocysteinemia (PHHC) on the neuronal migration, neural tissue maturation, and the expression of signaling molecules in the rat fetal brain were described. Maternal hyperhomocysteinemia was induced in female rats by per os administration of 0.15% aqueous methionine solution in the period of days 4-21 of pregnancy. Behavioral tests revealed a delay in PHHC male pups maturing. Ultrastructure of both cortical and hippocampus tissue demonstrated the features of the developmental delay. PHHC was shown to disturb both generation and radial migration of neuroblasts into the cortical plate. Elevated Bdnf expression, together with changes in proBDNF/mBDNF balance, might affect neuronal cell viability, positioning, and maturation in PHHC pups. Reduced Kdr gene expression and the content of SEMA3E might lead to impaired brain development. In the brain tissue of E20 PHHC fetuses, the content of the procaspase-8 was decreased, and the activity level of the caspase-3 was increased; this may indicate the development of apoptosis. PHHC disturbs the mechanisms of early brain development leading to a delay in brain tissue maturation and formation of the motor reaction of pups.

Theoretical Investigation of the Phenomenon of Space Charge Breakdown in Electromembrane Systems.

At present, it is customary to consider the overlimit operating modes of electromembrane systems to be effective, and electroconvection as the main mechanism of overlimiting transfer. The breakdown of the space charge is a negative, "destructive" phenomenon, since after the breakdown the size and number of electroconvective vortices are significantly reduced, which leads to a decrease in mass transfer. Therefore, electromembrane desalination processes must be carried out before space charge breakdown occurs. Thus, the actual problem arises of determining at which potential jumps a breakdown of the space charge occurs at a given concentration of the solution. Electromembrane systems are used for desalination at electrolyte solution concentrations ranging from 1 to 100 mol/m3. In a theoretical study of increasing the efficiency of the desalination process, mathematical modeling is used in the form of a boundary value problem for the system of Nernst-Planck and Poisson (NPP) equations, which refers to "hard" problems that are difficult to solve numerically. This is caused by the appearance of a small parameter at the derivative in the Poisson equation in a dimensionless form, and, correspondingly, a boundary layer at ion-exchange membranes, where concentrations and other characteristics of the desalination process change exponentially. It is for this reason that the numerical study of the boundary value problem is currently obtained for initial concentrations of the order of 0.01 mol/m3. The paper proposes a new numerical-analytical method for solving boundary value problems for the system of Nernst-Planck and Poisson equations for real initial concentrations, using which the phenomenon of space charge breakdown (SCB) in the cross section of the desalination channel in potentiostatic and potentiodynamic modes is studied. The main regularities of the appearance and interaction of charge waves, up to their destruction (breakdown), are established. A simple formula is proposed for engineering calculations of the potential jump depending on the concentration of the solution, at which the breakdown of the space charge begins.

Theoretical Analysis of Electroconvection in the Electrodialysis Desalination Channel under the Action of Direct Current.

The development of electroconvection in electromembrane systems is a factor that increases the efficiency of the electrolyte solution desalination process. The desalination of the solution, manifested by a change in the distribution of the ion concentration, can affect the mechanisms of development of electroconvection. The purpose of this work is to study the electroconvective flow developing in the desalination channel under various desalination scenarios. The study was carried out on the basis of a mathematical model of the transfer of binary electrolyte ions in the desalination channel formed between the anion and cation exchange membranes under the action of DC current. An analytical estimation of the threshold current density reflecting the conditions of the system transition into a quasi-stationary state has been obtained. The chronopotentiograms of the desalination channel and the thickness of the electroconvective mixing layer are calculated for both pre-threshold and supra-threshold values of the current density.

Changes in Metabotropic Glutamate Receptor Gene Expression in Rat Brain in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy.

Preventing epileptogenesis in people at risk is an unmet medical need. Metabotropic glutamate receptors (mGluRs) are promising targets for such therapy. However, drugs acting on mGluRs are not used in the clinic due to limited knowledge of the involvement of mGluRs in epileptogenesis. This study aimed to analyze the changes in gene expression of mGluR subtypes (1-5, 7, 8) in various rat brain regions in the latent and chronic phases of a lithium-pilocarpine model of epilepsy. For this study, multiplex test systems were selected and optimized to analyze mGluR gene expression using RT-qPCR. Region- and phase-specific changes in expression were revealed. During the latent phase, mGluR5 mRNA levels were increased in the dorsal and ventral hippocampus, and expression of group III genes was decreased in the hippocampus and temporal cortex, which could contribute to epileptogenesis. Most of the changes in expression detected in the latent stage were absent in the chronic stage, but mGluR8 mRNA production remained reduced in the hippocampus. Moreover, we found that gene expression of group II mGluRs was altered only in the chronic phase. The study deepened our understanding of the mechanisms of epileptogenesis and suggested that agonists of group III mGluRs are the most promising targets for preventing epilepsy.

Experimental and Theoretical Study of an Autowave Process in a Magnetic Fluid.

Magnetic fluid (MF) is a colloidal system consisting of ferromagnetic particles (magnetite) with a diameter of ~10 nm suspended in a dispersion medium of a carrier fluid (for example, kerosene). A distinctive feature of magnetic fluid is the fact that when an electric field is applied to it using two electrodes, thin layers consisting of close-packed particles of the dispersed phase are formed in the regions near the surface of both electrodes. These layers significantly affect the macroscopic properties of the colloidal system. In this work, the interpretation of the near-electrode layer is for the first time given as a new type of liquid membrane, in which the particles of the dispersed phase become charged with the opposite sign. On the basis of experimental studies, we propose a physicochemical mechanism of the autowave process in a cell with a magnetic fluid. It is based on the idea of oppositely recharging colloidal particles of magnetite in a liquid membrane. A mathematical model of an autowave process, which is described by a system of coupled partial differential equations of Nernst-Planck-Poisson and Navier-Stokes with appropriate boundary conditions, is proposed for the first time. One-dimensional, two-dimensional, and three-dimensional versions of the model are considered. The dependence of the frequency of concentration fluctuations on the stationary voltage between the electrodes was obtained, and the time of formation of a liquid membrane was estimated. Qualitative agreement between theoretical and experimental results has been established.

MTEP, a Selective mGluR5 Antagonist, Had a Neuroprotective Effect but Did Not Prevent the Development of Spontaneous Recurrent Seizures and Behavioral Comorbidities in the Rat Lithium-Pilocarpine Model of Epilepsy.

Metabotropic glutamate receptors (mGluRs) are expressed predominantly on neurons and glial cells and are involved in the modulation of a wide range of signal transduction cascades. Therefore, different subtypes of mGluRs are considered a promising target for the treatment of various brain diseases. Previous studies have demonstrated the seizure-induced upregulation of mGluR5; however, its functional significance is still unclear. In the present study, we aimed to clarify the effect of treatment with the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that the administration of MTEP during the latent phase of the model did not improve survival, prevent the development of epilepsy, or attenuate its manifestations in rats. However, MTEP treatment completely prevented neuronal loss and partially attenuated astrogliosis in the hippocampus. An increase in excitatory amino acid transporter 2 expression, which has been detected in treated rats, may prevent excitotoxicity and be a potential mechanism of neuroprotection. We also found that MTEP administration did not prevent the behavioral comorbidities such as depressive-like behavior, motor hyperactivity, reduction of exploratory behavior, and cognitive impairments typical in the lithium-pilocarpine model. Thus, despite the distinct neuroprotective effect, the MTEP treatment was ineffective in preventing epilepsy.

Maternal Hyperhomocysteinemia Produces Memory Deficits Associated with Impairment of Long-Term Synaptic Plasticity in Young Rats.

Maternal hyperhomocysteinemia (HCY) is a common pregnancy complication caused by high levels of the homocysteine in maternal and fetal blood, which leads to the alterations of the cognitive functions, including learning and memory. In the present study, we investigated the mechanisms of these alterations in a rat model of maternal HCY. The behavioral tests confirmed the memory impairments in young and adult rats following the prenatal HCY exposure. Field potential recordings in hippocampal slices demonstrated that the long-term potentiation (LTP) was significantly reduced in HCY rats. The whole-cell patch-clamp recordings in hippocampal slices demonstrated that the magnitude of NMDA receptor-mediated currents did not change while their desensitization decreased in HCY rats. No significant alterations of glutamate receptor subunit expression except GluN1 were detected in the hippocampus of HCY rats using the quantitative real-time PCR and Western blot methods. The immunofluorescence microscopy revealed that the number of synaptopodin-positive spines is reduced, while the analysis of the ultrastructure of hippocampus using the electron microscopy revealed the indications of delayed hippocampal maturation in young HCY rats. Thus, the obtained results suggest that maternal HCY disturbs the maturation of hippocampus during the first month of life, which disrupts LTP formation and causes memory impairments.

Early Life Febrile Seizures Impair Hippocampal Synaptic Plasticity in Young Rats.

Febrile seizures (FSs) in early life are significant risk factors of neurological disorders and cognitive impairment in later life. However, existing data about the impact of FSs on the developing brain are conflicting. We aimed to investigate morphological and functional changes in the hippocampus of young rats exposed to hyperthermia-induced seizures at postnatal day 10. We found that FSs led to a slight morphological disturbance. The cell numbers decreased by 10% in the CA1 and hilus but did not reduce in the CA3 or dentate gyrus areas. In contrast, functional impairments were robust. Long-term potentiation (LTP) in CA3-CA1 synapses was strongly reduced, which we attribute to the insufficient activity of N-methyl-D-aspartate receptors (NMDARs). Using whole-cell recordings, we found higher desensitization of NMDAR currents in the FS group. Since the desensitization of NMDARs depends on subunit composition, we analyzed NMDAR current decays and gene expression of subunits, which revealed no differences between control and FS rats. We suggest that an increased desensitization is due to insufficient activation of the glycine site of NMDARs, as the application of D-serine, the glycine site agonist, allows the restoration of LTP to a control value. Our results reveal a new molecular mechanism of FS impact on the developing brain.

The application of the self-probing primer PCR for quantitative expression analysis of R607Q (un)edited GluA2 AMPA receptor mRNA.

Adenosine deaminase-dependent RNA editing is a widespread universal mechanism of posttranscriptional gene function modulation. Changes in RNA editing level may contribute to various physiological and pathological processes. In the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor GluA2 subunit, A-I editing in the Q607R site leads to dramatic changes in function, making the receptor channel calcium-impermeable. A standard approach for quantifying (un)edited RNAs is based on endpoint PCR (Sanger sequencing or restriction analysis), a time-consuming and semiquantitative method. We aimed to develop RT-qPCR assays to quantify rat Q607R (A-I) edited/unedited mRNA in samples in the present work. Based on self-probing PCR detection chemistry, described initially for detecting short DNA fragments, we designed and optimised RT-qPCR assays to quantify Q607R (un)edited mRNA. We used self-probing primer PCR technology for mRNA quantification for the first time. Using a novel assay, we confirmed that Q607R GluA2 mRNA editing was increased in 14-day- (P14) or 21-day-old (P21) postnatal brain tissue (hippocampus) compared to the embryonic brain (whole brains at E20) in Wistar rats. Q607R unedited GluA2 mRNA was detectable by our assay in the cDNA of mature brain tissue compared to that derived through classical methods. Thus, self-probing primer PCR detection chemistry is an easy-to-use approach for RT-qPCR analysis of RNA editing.

Alterations in mRNA and Protein Expression of Glutamate Receptor Subunits Following Pentylenetetrazole-induced Acute Seizures in Young Rats.

Acute seizures can severely affect brain function and development. However, the underlying pathophysiological mechanisms are still poorly understood. Disturbances of the glutamatergic system are considered one of the critical mechanisms of neurological abnormalities. In the present study, we analyzed changes in the expression of NMDA and AMPA receptor subunits in the different brain regions (dorsal hippocampus, amygdala, and the medial prefrontal, temporal, and entorhinal cortex) using a pentylenetetrazole (PTZ) model of seizures in 3-week-old rats. A distinctive feature of this model is that the administration of PTZ causes severe acute seizures, which are not followed by the development of spontaneous recurrent seizures later on. Subunit expression was analyzed using qRT-PCR and Western blotting during the first week after seizures. The most pronounced alterations of mRNA and protein levels were observed in the dorsal hippocampus. We found decreased expression of the GluA2 mRNA 7 days after seizures (PSE7), as well as reduced GluN2a protein levels on PSE7. Significant alterations in the expression of different receptor subunits in the mRNA but not protein levels were observed in the entorhinal cortex and amygdala. In contrast, in the medial prefrontal and temporal cortex, we found almost no changes in the expression of the studied genes. The identified changes deepen our understanding of post-seizure disturbances in the developing brain and confirm that although various brain structures are involved in seizures, the hippocampus is the most vulnerable.

Anakinra Reduces Epileptogenesis, Provides Neuroprotection, and Attenuates Behavioral Impairments in Rats in the Lithium-Pilocarpine Model of Epilepsy.

Temporal lobe epilepsy is a widespread chronic disorder that manifests as spontaneous seizures and is often characterized by refractoriness to drug treatment. Temporal lobe epilepsy can be caused by a primary brain injury; therefore, the prevention of epileptogenesis after a primary event is considered one of the best treatment options. However, a preventive treatment for epilepsy still does not exist. Neuroinflammation is directly involved in epileptogenesis and neurodegeneration, leading to the epileptic condition and cognitive decline. In the present study, we aimed to clarify the effect of treatment with a recombinant form of the Interleukin-1 receptor antagonist (anakinra) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that anakinra administration during the latent phase of the model significantly suppressed the duration and frequency of spontaneous recurrent seizures in the chronic phase. Moreover, anakinra administration prevented some behavioral impairments, including motor hyperactivity and disturbances in social interactions, during both the latent and chronic periods. Histological analysis revealed that anakinra administration decreased neuronal loss in the CA1 and CA3 areas of the hippocampus but did not prevent astro- and microgliosis. The treatment increased the expression level of the solute carrier family 1 member 2 gene (Slc1a2, encoding excitatory amino acid transporter 2 (EAAT2)) in the hippocampus, potentially leading to a neuroprotective effect. However, the increased gene expression of proinflammatory cytokine genes (Interleukin-1ß (Il1b) and tumor necrosis factor α (Tnfa)) and astroglial marker genes (glial fibrillary acidic protein (Gfap) and inositol 1,4,5-trisphosphate receptor type 2 (Itpr2)) in experimental rats was not affected by anakinra treatment. Thus, our data demonstrate that the administration of anakinra during epileptogenesis has some beneficial disease-modifying effects.