High and Low-Frequency Stimulation Effect on Epileptiform Activity in Brain Slices.

Background: Neurostimulation is one of the new therapeutic approaches in patients with drug-resistant epilepsy, and despite its high efficiency, its mechanism of action is still unclear. On the one hand, electrical stimulation in the human brain is immoral; on the other hand, the creation of the epilepsy model in laboratory animals affects the entire brain network. As a result, one of the ways to achieve the neurostimulation mechanism is to use epileptiform activity models In vitro. In vitro models, by accessing the local network from the whole brain, we can understand the mechanisms of action of neurostimulation. Methods: A literature search using scientific databases including PubMed, Google Scholar, and Scopus, using "Neurostimulation" and "epileptiform activity" combined with "high-frequency stimulation", " low-frequency stimulation ", and "brain slices" as keywords were conducted, related concepts to the topic gathered and are used in this paper. Results: Electrical stimulation causes neuronal depolarization and the release of GABAA, which inhibits neuronal firing. Also, electrical stimulation inhibits the nervous tissue downstream of the stimulation site by preventing the passage of nervous activity from the upstream to the downstream of the axon. Conclusion: Neurostimulation techniques consisting of LFS and HFS have a potential role in treating epileptiform activity, with some studies having positive results. Further investigations with larger sample sizes and standardized outcome measures can be conducted to validate the results of previous studies.

The plasma level of glutamic acid decarboxylase 65 (GAD65) increased in severely autistic Iranian children.

INTRODUCTION: Autism spectrum disorder (ASD) is a neurodevelopmental disorder. The major etiological mechanism lies in glutamatergic/GABAergic imbalance. The aim of this study was to evaluate the plasma levels of glutamic acid decarboxylase 65 (GAD65) protein in mildly and severely autistic patients, and also to compare plasma GAD65 concentration in mild and severe autism. METHOD: In total, 62 autistic patients (aged 6-9 years) and 17 age-matched neurotypically healthy controls were included in the study. The diagnosis, as well as the level of autism, was assessed by applying the Gilliam Autism Rating Scale. Plasma GAD65 protein level was determined using an enzyme-linked immunosorbent assay (ELISA) kit for GAD65. RESULTS: Our findings showed no remarkable alteration in plasma GAD65 concentration in patients with mild autism as compared to healthy subjects, while patients with severe autism showed an increased plasma level of GAD65 as compared to healthy controls and mildly autistic patients. CONCLUSION: Our findings suggest the level of plasma GAD65 to be considered a potential diagnostic biomarker for the severity of autism (Fig. 2, Ref. 40).

Exercise Improved the Anti-Epileptic Effect of Carbamazepine through GABA Enhancement in Epileptic Rats.

Carbamazepine (CBZ) is an anticonvulsant drug that usually is used for the treatment of seizures. The anti-epileptic and the anti-epileptogenic effect of exercise has been reported, as well. This study was aimed to evaluate the synergic effect of combined therapy of exercise and CBZ in epileptic rats, as well as the alternation of the GABA pathway as a possible involved mechanism. The seizure was induced by pentylenetetrazol (PTZ) injection. Animals were divided into sham, seizure, exercise (EX), CBZ (25, 50 and 75), EX + CBZ (25, 50 and 75). Treadmill forced running for 30 min has been considered as the exercise 5 days per week for four weeks. CBZ was injected in doses of 25, 50 and 75 mg/kg, half an hour before seizure induction and 5 h after doing exercise in the animals forced to exercise. Seizure severity reduced and latency increased in the EX + CBZ (25) and EX + CBZ (50) groups compared to the seizure group. The distribution of GAD65 in both hippocampal CA1 and CA3 areas increased in the EX + CBZ (75) group. GABAA receptor α1 was up-regulated in the CA3 area of the EX + CBZ (75) group. The distribution of GAD65 in the cortical area increased in EX, EX + CBZ (50), CBZ (75) and EX + CBZ (75) groups. GABAA receptor α1 was up-regulated in the neocortex of EX + CBZ (50), CBZ (75) and EX + CBZ (75) groups. Our findings suggested that exercise has improved the efficacy of CBZ and reduced the anti-epileptic dose. The enhancement of GABA signaling might be involved in the synergistic effect of exercise and CBZ.

Discrepancies of Notch 1 receptor during development of chronic seizures.

The critical role of Notch signaling has been shown in the pathogenesis of some neurological disorders including schizophrenia, epilepsy and Alzheimer's disease. This study was aimed to evaluate the role of Notch 1 receptor in epileptogenesis as well as seizure characteristics. The animals were divided into three groups of sham, early stage and end stage. In sham group: Normal saline was injected intraperitoneally (ip) in the same as protocol of pentylenetetrazol (PTZ) injection. PTZ was injected (ip) every 48 hr over a period of 1 week in the group of early stage and over a period of 4 weeks in the end stage. The gene expression as well as distribution of Notch 1 receptor was assessed in the parietal cortex and hippocampus. In addition, the effect of agonist or antagonist of Notch 1 receptor was assessed on the epileptic discharges induced by PTZ injection. The gene expression of Notch 1 decreased in the hippocampus significantly in the end-stage group compared with sham, and early groups. Furthermore, distribution of Notch 1 receptor increased in the somatosensory cortex and decreased in the CA1 hippocampal area in the end-stage group. Intraventricular microinjection of Notch 1 agonist significantly increased the amplitude as well as frequency of spikes and decreased the latency of first epileptic discharges. Our findings illustrate the critical role of Notch signalling as a potential pathway in the epileptogenesis during development of chronic seizures.

The organizational culture of Iran University of Medical Sciences from the viewpoint of faculty members.

Background: It has been widely acknowledged that change and constant modification is the key to survive for any organization among their rivals. Since success in implementing changes in the organization strongly depends on the organizational culture, this study aims to assess the organizational culture in Iran University of Medical Sciences. The results of this study can be beneficial in initiating a movement towards the third - and fourth generation of universities. Methods: This study is descriptive-correlational. The Organizational Culture Assessment Instrument (OCAI) was employed to collect data. A questionnaire was sent to the faculty members via email, and the responses were collected and analyzed. Results: Out of the 982 faculty members, 189 participated (20.7%) in this study. Analysis showed that the organizational culture of the university is congruent and harmonious and in the current state, it is primarily hierarchical (31%) and market-oriented (28%) with emphasis on stability and control in the organization. Whereas, faculty members tend to move the organizational culture of the university towards adhocracy (30%) and clan culture (29%). Conclusion: University administrators must strengthen the culture of innovation and creativity based on the needs of the market. This only can be achieved by supporting teamwork in their move towards desired change.

Effects of garlic extract on spreading depression: In vitro and in vivo investigations.

OBJECTIVES: The potential use of garlic for prevention and treatment of different types of headaches has been suggested by several medieval literatures. Cortical spreading depression (CSD), a propagating wave of neuroglial depolarization, was established as a target for anti-migraine drugs. This study was designed to investigate the effect of garlic extract on CSD in adult rats. METHODS: CSD was induced by KCl microinjection in the somatosensory cortex. The effects of five different concentrations of garlic oil (1-500 µl/l) were tested on different characteristic features of CSD in necocortical slices. In in vivo experiments, the effects of garlic oil on electrophysiological and morphological changes induced by CSD were investigated. RESULTS: Garlic oil in a dose-dependent manner decreased the amplitude of CSD but not its duration and velocity in neocortical brain slices. Garlic oil at concentration of 500 µl/l reversibly reduced the amplitude of the field excitatory post-synaptic potentials and inhibited induction of long-term potentiation in the third layer of neocortical slices. In in vivo studies, systemic application of garlic oil (1 ml/l) for three consecutive days reduced the amplitude and repetition rate of CSD. Garlic oil also prevented of CSD-induced reactive astrocytosis in the neocortex. DISCUSSION: Garlic oil suppresses CSD, likely via inhibition of synaptic plasticity, and prevents its harmful effects on astrocyte. Further studies are required to identify the exact active ingredient(s) of garlic oil that inhibit CSD and may have the potential to use in treatment of CSD-related disorders.

Curcumin Lowers the Accelerated Speed of Epileptogenesis by Traumatic Brain Injury.

Background: Traumatic brain injury or TBI can underlie epilepsy. Prevention of PTE has been of great interest to scientists. Given the antiepileptic, antioxidant and anti-inflammatory activities of curcumin, we examined whether this compound can affect epileptogenesis in rats after TBI. Methods: Curcumin was injected once a day for two weeks. TBI was induced in the temporal cortex of anesthetized rats using a controlled cortical impact device. One day after TBI, pentylenetetrazole (PTZ), 35 mg/kg, was injected i.p. every other day until manifestation of generalized seizures. The number of PTZ injections was then recorded. Moreover, the extent of cortical and hippocampal IL-1ß and glial fibrillary acidic protein (GFAP) expression in the epileptic rats were measured by Western blot analysis. Results: Curcumin 50 and 150 mg/kg prevented the development of kindling, whereas TBI accelerated the rate of kindling. Curcumin 20 mg/kg prohibited kindling facilitation by TBI, and reduced the expression of IL-1ß and GFAP induced by TBI. Conclusion: Curcumin can stop the acceleration of epileptogenesis after TBI in rats. Inhibiting hippocampal and cortical overexpression of IL-1ß and GFAP seems to be involved in this activity.

Diminution of the NMDA receptor NR2B subunit in cortical and subcortical areas of WAG/Rij rats.

Modulation of glutamatergic NMDA receptors affects the synchronization of spike discharges in in WAG/Rij rats, a valid genetic animal model of absence epilepsy. In this study, we describe the alteration of NR2B subunit of NMDA receptors expression in WAG/Rij rats in different somatosensory cortical layers and in hippocampal CA1 area. Experimental groups were divided into four groups of six rats of both WAG/Rij and Wistar strains with 2 and 6 months of age. The distribution of NR2B receptors was assessed by immunohistochemical staining in WAG/Rij and compared with age-matched Wistar rats. The expression of NR2B subunit was significantly decreased in different somatosensory cortical layers in 2- and 6-month-old WAG/Rij rats. In addition, the distribution of NR2B in hippocampal CA1 area was lower in 6-month-old WAG/Rij compared with age-matched Wistar rats. The reduction of NR2B receptors in different brain areas points to disturbance of glutamate receptors expression in cortical and subcortical areas in WAG/Rij rats. An altered subunit assembly of NMDA receptors may underlie cortical hyperexcitability in absence epilepsy.

Behavioural and histopathological assessment of the effects of periodic fasting on pentylenetetrazol-induced seizures in rats.

OBJECTIVES: Periodic fasting (PF) was suggested to display antiepileptic and neuroprotective effects, which is in stark contrast to severe fasting or starvation. However, these beneficial effects seem to depend on the type and duration of the used feeding protocol. There are discrepancies concerning both antiepileptic and neuroprotective effects of a PF-diet during repetitive seizures in different epilepsy models. This study was designed to evaluate the effects of different PF protocols on behavioural and histopathological consequences of epilepsy in adult rats. METHODS: Recurrent generalized seizures were caused by repetitive injection of pentylenetetrazol (PTZ) for a period of 4 weeks every other day. While control animals had free access to food and water, animals on a PF-diet were on intermittent fasting for 24 hours every 48 hours for 4 weeks before (T1), after (T2), or both before and after (T3) the injection of PTZ. Behavioural studies were carried out after PTZ injections and histological investigations were performed after the experiments were completed. RESULTS: Seizure assessment showed that the severity of seizures was significantly decreased in groups T1 and T3 when compared with control rats. Dark neuron densities in hippocampal CA1 and CA3 areas were decreased in PF groups, but never in the temporal cortex. The PF-diet also decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling-positive neurons in the hippocampus in both areas and all PF-diet protocols. DISCUSSION: These results support the idea that a PF-diet has anticonvulsive and neuroprotective effects on epileptic rats but underlines that different PF-diet protocols can have varying effects. Anticonvulsive effects were strongest when the PF-diet started before the onset of excitotoxic injuries, the number of dark neurons was decreased and apoptosis was prevented by all PF-diet protocols investigated in this work. Further evaluation of PF-diet protocols for possible clinical anticonvulsant and neuroprotective effects is suggested.

Anticonvulsant and neuroprotective effects of Pimpinella anisum in rat brain.

BACKGROUND: Essential oil of Pimpinella anisum L. Apiaceae (anise oil) has been widely used in traditional Persian medicine to treat a variety of diseases, including some neurological disorders. This study was aimed to test the possible anti-seizure and anti-hypoxia effects of anise oil. METHODS: The effects of different concentrations of anise oil were tested on seizure attacks induced by pentylenetetrazol (PTZ) injection and neuronal hypoxia induced by oxygen withdrawal as well as on production of dark neurons and induction of long-term potentiation (LTP) in in vivo and in vitro experimental models of rat brain. RESULTS: Anise oil significantly prolonged the latency of seizure attacks and reduced the amplitude and duration of epileptiform burst discharges induced by injection of intraperitoneal PTZ. In addition, anise oil significantly inhibited production of dark neurons in different regions of the brain in epileptic rats. Anise oil also significantly enhanced the duration of the appearance of anoxic terminal negativity induced by oxygen withdrawal and inhibited induction of LTP in hippocampal slices. CONCLUSIONS: Our data indicate the anticonvulsant and neuroprotective effects of anise oil, likely via inhibition of synaptic plasticity. Further evaluation of anise oil to use in the treatment of neurological disorders is suggested.

Alteration in Neuregulin 1/ERbB4 in Absence Epilepsy: Regulatory Effect on TRPV1 Expression.

Introduction: The footprint of Neuregulin 1 (NRG1) / ERbB4 in the pathophysiology of some neurological disorders and TRPV1 regulation has been indicated. The alterations in NRG1 and ErbB4 as well as the TRPV1 signaling pathway were investigated during the development of absence epilepsy in the genetic animal model of absence epilepsy. Methods: Male WAG/Rij and Wistar rats were divided into four experimental groups of two and six months of age. The protein levels of NRG1, ERbB4, and TRPV1 were measured in the somatosensory cortex and hippocampus. Results: The cortical protein levels of NRG1 and ErbB4 in the 6-month-old WAG/Rij rats were lower than in Wistar rats. Protein levels of TRPV1 were lower in two- and six-month-old WAG/Rij rats compared to age-matched Wistar rats.Hippocampal protein levels of NRG1 in 6-month-old WAG/Rij rats were lower than two-month-old WAG/Rij rats. Low levels of ErbB4 protein in two-month-old and high levels in six-month-old WAG/Rij rats were found compared to Wistar rats. Protein levels of TRPV1 were lower in the two-month-old and higher in the six-month-old WAG/Rij rats compared to age-matched Wistar rats.Furthermore, a high correlation between NRG1/ERbB4 and TRPV1 expressions in the cortex and hippocampus was indicated. The expression of NRG1/ERbB4 and TRPV1 followed a similar pattern during the life span of Wistar and WAG/Rij rats. Conclusion: Our findings indicated the potential role of the NRG1/ErbB4 pathway as well as TRPV1 in the pathogenesis of absence epilepsy. The regulatory effect of the ERbB4 receptor on the TRPV1 expression has been suggested following the similar pattern of expression.

Melittin ameliorates motor function and prevents autophagy-induced cell death and astrogliosis in rat models of cerebellar ataxia induced by 3-acetylpyridine.

BACKGROUND: Cerebellar ataxia (CA) is a form of ataxia that adversely affects the cerebellum. This study aims to investigate the therapeutic effects of melittin (MEL) on a 3-acetylpyridine-induced (3-AP) cerebellar ataxia (CA) rat model. METHODS: Initially, CA rat models were generated by 3-AP administration followed by the subcutaneous injection of MEL. The open-field test was used for the evaluation of locomotion and anxiety. Immunohistochemistry was also conducted for the autophagy markers of LC3 and Beclin1. In the next step, the morphology of the astrocyte, the cell responsible for maintaining homeostasis in the CNS, was evaluated by the Sholl analysis. RESULTS: The findings suggested that the administration of MEL in a 3-AP model of ataxia improved locomotion and anxiety (P < 0.001), decreased the expression of LC3 (P < 0.01) and Beclin1 (P < 0.05), increased astrocyte complexity (P < 0.05) and reduced astrocyte cell soma size (P < 0.001). CONCLUSIONS: Overall, the findings imply that the MEL attenuates the 3-AP-induced autophagy, causes cell death and improves motor function. As such, it could be used as a therapeutic procedure for CA due to its neuroprotective effects.

Evaluation of Serum Levels of Transient Receptor Potential Cation Channel Subfamily V Member 1, Vasoactive Intestinal Polypeptide, and Pituitary Adenylate Cyclase-Activating Polypeptide in Chronic and Episodic Migraine: The Possible Role in Migraine Transformation.

Objectives: This study aimed to investigate the role of serum levels of transient receptor potential cation channel subfamily V member 1 (TRPV1), vasoacive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) in the development and also the transformation of migraine in patients suffering from migraine. Methods: Eighty-nine participants with a mean age of 39 years were divided into 23 episodic migraine (EM), 36 chronic migraine (CM), and 30 healthy control groups. Demographic, anthropometric, and headache characteristic information, and also blood samples, was collected. Serum levels of TRPV1, VIP, and PACAP were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Results: Based on our findings, the serum level of TRPV1 was significantly higher in CM compared to the control group (p < 0.05), whereas serum levels of VIP (p < 0.01) and PACAP (p < 0.05) in the EM group were significantly more than the control group. There was no significant difference between EM and CM groups. Conclusions: An elevation in the serum levels of TRVP1 among chronic migraineurs and increments in the levels of VIP and PACAP were observed among EM patients compared to healthy subjects. However, our data failed to demonstrate the probable role of these biomarkers in migraine progression, and more studies are needed to clarify the molecular mechanisms involved in migraine progression.

Olfactory mucosa stem cells delivery via nasal route: a simple way for the treatment of Parkinson disease.

Finding a simple and effective way for transferring cells to the brain lesion site with minimum side effects mounts a challenge in cell therapy. Cell delivery via nasal route using the bypassing the blood-brain barrier (BBB) property is a simple and non-invasive strategy without serious complications such as trauma. Therefore, it is a suitable technique to treat neurodegenerative disorders like Parkinson's disease (PD). Olfactory ectomesenchymal stem cells (OE-MSCs) located in the lamina propria of olfactory mucosa could be differentiated into dopaminergic neurons under in vitro and in vivo conditions. Thus, OE-MSCs represent a good source of Parkinson's stem cell-based therapy. In this research, we studied thirty male rats (n = 10 in each group) in three control (Ctl), lesion (LE), and intranasal administration (INA) groups to investigate the therapeutic effect of intranasal injection of OE-MSCs in the Parkinson's animal models. To do so, we examined the homing variation of OE-MSCs in different brain regions such as olfactory bulb (OB), cortex, striatum (Str), hippocampus (HPC), and substantia nigra (SN). The results of real-time PCR and immunohistochemistry (IHC) analysis showed the expression of dopaminergic neuron markers such as PITX3, PAX2, PAX5 (as dopaminergic neurons markers), tyrosine hydroxylase (TH), and dopamine transporter (DAT) 2 months after INA of 1 × 106 OE-MSCs. The results confirmed that IN OE-MSCs delivery into the central nervous system (CNS) was powerful enough to improve the behavioral functions in the animal models of PD.

Experimental Models of Absence Epilepsy.

INTRODUCTION: Absence epilepsy is a brief non-convulsive seizure associated with sudden abruptness in consciousness. Because of the unpredictable occurrence of absence seizures and the ethical issues of human investigation on the pathogenesis and drug assessment, researchers tend to study animal models. This paper aims to review the advantages and disadvantages of several animal models of nonconvulsive induced seizure. METHODS: The articles that were published since 1990 were assessed. The publications that used genetic animals were analyzed, too. Besides, we reviewed possible application methods of each model, clinical types of seizures induced, purposed mechanism of epileptogenesis, their validity, and relevance to the absence epileptic patients. RESULTS: The number of studies that used genetic models of absence epilepsy from years of 2000 was noticeably more than pharmacological models. Genetic animal models have a close correlation of electroencephalogram features and epileptic behaviors to the human condition. CONCLUSION: The validity of genetic models of absence epilepsy would motivate the researchers to focus on genetic modes in their studies. As there are some differences in the pathophysiology of absence epilepsy between animal models and humans, the development of new animal models is necessary to understand better the epileptogenic process and, or discover novel therapies for this disorder.

Glutamate Signaling Pathway in Absence Epilepsy: Possible Role of Ionotropic AMPA Glutamate Receptor Type 1 Subunit.

AMPA receptors, consisting of glutamate receptor type1 (GluR1) subunit are involved in the pathophysiology of some neurological disorders. In this study, the role of the GluR1 subunit in the development, as well as features of absence seizures were assessed. Both Wistar and WAG/Rij (a genetic animal model of absence epilepsy) rats with 2 and 6-month ages were included in the study. The expression of GluR1 was measured in the somatosensory cortex. Moreover, the effects of pharmacological activation and inhibition of AMPA receptors on the characteristic of absence epileptic activities were evaluated by microinjection of agonist or antagonist of AMPA receptors on the somatosensory cortex in the epileptic WAG/Rij rats. Distribution of the GluR1 subunit of AMPA receptors in the both IV (p < 0.001) and VI (p < 0.01) layers of the somatosensory cortex in the epileptic WAG/Rij rats was higher than non-epileptic animals. In addition, the microinjection of AMPA receptors agonist on the somatosensory cortex of the WAG/Rij rats increased both amplitude (p < 0.01) and duration (p < 0.001) of spike-wave discharges (SWDs), while injection of antagonist reduced amplitude (p < 0.001) and duration (p < 0.01) of SWDs in the somatosensory cortex of epileptic rats. The high expression of GluR1 in the somatosensory cortex of epileptic rats suggests the role of AMPA receptors consisting of the GluR1 subunit in the development of absence seizures. The modulatory effects AMPA receptors on the feature of SWDs suggest the potential of AMPA receptors antagonists as a therapeutic target for absence epilepsy.

The effect of exercise on GABA signaling pathway in the model of chemically induced seizures.

AIMS: Gamma amino butyric acid (GABA) imbalance plays a critical role in most neurological disorders including epilepsy. This study assessed the involvement of mild exercise on GABA imbalance following by seizure induction in rats. MAIN METHODS: Seizure was induced by pentylentetrazole (PTZ) injection. Animals were divided into sham, seizure, exercise (EX), co-seizure-induced exercise (Co-SI EX) and Pre-SI EX groups. In the Co-SI EX group, doing exercise and seizure induction was carried out during four weeks. Animals in the Pre-SI EX group exercised in week 1 to week 8 and seizures were induced in week 5 to week 8. Seizure properties, neural viability and expressions of glutamic acid decarboxylase 65 (GAD65) and GABAA receptor α1 in the hippocampus were assessed. KEY FINDINGS: Seizure severity reduced and latency increased in the Co-SI EX and Pre-SI EX groups compared to seizure group. The mean number of dark neurons decreased in all exercise groups compared to seizure group in both CA1 and CA3 areas. The gene level of GAD65 and GABAA receptor α1 was highly expressed in the Co-SI EX group in the hippocampal area. Distribution of GAD65 in the both CA1 and CA3 areas increased in the EX and Co-SI EX groups. GABAA receptor α1 was up-regulated in the CA3 area of Co-SI EX group and down-regulated in the CA1 and CA3 areas of Pre-SI EX group. SIGNIFICANCE: These findings suggest that exercise develop anti-epileptic as well as neuroprotective effects by modulating of GABA disinhibition.

Stable Knockdown of Adenosine Kinase by Lentiviral Anti-ADK miR-shRNAs in Wharton's Jelly Stem Cells.

OBJECTIVES: In this study, we describe an efficient approach for stable knockdown of adenosine kinase (ADK) using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells (hWJMSCs). These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy. MATERIALS AND METHODS: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA (shRNAs). First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction (qRT-PCR) was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker (CD44+) and lack of expression of hematopoietic lineage marker (CD45-). Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs. RESULTS: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miRshRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miRshRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level. CONCLUSIONS: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors (AAV) might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy.

Developmental changes in Notch1 and NLE1 expression in a genetic model of absence epilepsy.

Childhood absence epilepsy (CAE) is an epilepsy syndrome with seizures occurring in the early childhood, highlighting that seizures susceptibility in CAE is dependent on brain development. The Notch 1 signalling pathway is important in brain development, yet the role of the Notch1 signalling pathway in CAE remains elusive. We here explored Notch1 and its modulator notchless homologue 1 (NLE1) expression in WAG/Rij and control rats using immunohistochemistry. Functional Notch 1 effects were assessed in WAG/Rij rats in vivo. WAG/Rij rats lack the developmental increase in cortical Notch1 and NLE 1 mRNA expression seen in controls, and Notch 1 and NLE1 mRNA and protein expression were lower in somatosensory cortices of WAG/Rij rats when compared to controls. This coincided with an overall decreased cortical GFAP expression in the early development in WAG/Rij rats. These effects were region-specific as they were not observed in thalamic tissues. Neuron-to-glia ratio as a marker of the impact of Notch signalling on differentiation was higher in layer 4 of somatosensory cortex of WAG/Rij rats. Acute application of Notch 1 agonist Jagged 1 suppressed, whereas DAPT, a Notch antagonist, facilitated spike and wave discharges (SWDs) in WAG/Rij rats. These findings point to Notch1 as an important signalling pathway in CAE which likely shapes architectural organization of the somatosensory cortex, a region critically involved in developmental epileptogenesis in CAE. More immediate effects of Notch 1 signalling are seen on in vivo SWDs in CAE, pointing to the Notch 1 pathway as a possible treatment target in CAE.