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- Is peripheral immunity regulated by blood-brain barrier permeability changes? [Journal Article]
- PLoS One 2014; 9(7):e101477.
S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses.
- The histone lysine demethylase Kdm6b is required for activity-dependent preconditioning of hippocampal neuronal survival. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Enzymes that regulate histone lysine methylation play important roles in neuronal differentiation, but little is known about their contributions to activity-regulated gene transcription in differentiated neurons. We characterized activity-regulated expression of lysine demethylases and lysine methyltransferases in the hippocampus of adult male mice following pilocarpine-induced seizure. Pilocarpine drove a 20-fold increase in mRNA encoding the histone H3 lysine27-specific demethylase Kdm6b selectively in granule neurons of the dentate gyrus, and this induction was recapitulated in cultured hippocampal neurons by bicuculline and 4-aminopyridine (Bic+4AP) stimulation of synaptic activity. Because activity-regulated gene expression is highly correlated with neuronal survival, we tested the requirement for Kdm6b expression in Bic+4AP induced preconditioning of neuronal survival. Prior exposure to Bic+4AP promoted neuronal survival in control neurons upon growth factor withdrawal, however this effect was ablated when we knocked down Kdm6b expression. Loss of Kdm6b did not disrupt activity-induced expression of most genes, including that of a gene set previously established to promote neuronal survival in this assay. However using bioinformatic analysis of RNA sequencing data, we discovered that Kdm6b knockdown neurons showed impaired inducibility of a discrete set of genes annotated for their function in inflammation. These data reveal a novel function for Kdm6b in activity-regulated neuronal survival, and they suggest that activity- and Kdm6b-dependent regulation of inflammatory gene pathways may serve as an adaptive pro-survival response to increased neuronal activity.
- Pharmacologically and Edinger-Westphal Stimulated Accommodation in Rhesus Monkeys does not Rely on Changes in Anterior Chamber Pressure. [JOURNAL ARTICLE]
- Exp Eye Res 2014 Jun 28.
This study was undertaken to understand the role of anterior chamber pressure (ACP) during pharmacological and Edinger-Westphal (EW) stimulated accommodation in anesthetized monkeys. Experiments were performed on one iridectomized eye each of 7 anesthetized adolescent rhesus monkeys. Accommodation was induced by EW stimulation (n=2) and intravenous administration of 0.25-4.0 mg/kg pilocarpine (n=6). Accommodative refractive and biometric changes were measured with continuous 60 Hz infrared photorefraction (n=6) and 100 Hz A-scan ultrasound biometry (n=1). An ocular perfusion system was used to measure and manipulate ACP. Pressure was recorded via a 27-gauge needle in the anterior chamber connected to a pressure transducer (n=7). The needle was also connected to a fluid reservoir to allow ACP to be manipulated and clamped (n=4) by raising or lowering the fluid reservoir. In all six pharmacologically stimulated monkeys ACP increased during accommodation, from 0.70 to 2.38 mmHg, four of which showed pressure decreases preceding the pressure increases. Two eyes also showed increases in ACP during EW-stimulated accommodation of 2.8 and 7.2 mmHg. ACP increased with increasing EW stimulus amplitudes (n=2). Clamping or externally manipulating ACP had no effect on resting refraction or on EW and pharmacologically stimulated accommodation in four eyes. The results show that EW stimulated and pharmacologically stimulated accommodation do not rely on ACP in rhesus monkeys.
- Effect of Androsterone after Pilocarpine-induced Status Epilepticus in Mice. [Journal Article]
- J Epilepsy Res 2014 Jun; 4(1):7-13.
Neurosteroids exert their antiepileptic effects via GABAA and NMDA receptors. Another cell death mechanism is excessive Ca(2+) influx into cells. Calbindin-D28k (CB) is a protein that modulates intracellular Ca(2+) in the nervous system. We evaluated whether androsterone up-regulates the expression of CB and has a neuroprotective effect by controlling Ca(2+) after pilocarpine-induced status epilepticus (SE) in mice.SE was induced in ICR mice by injection of pilocarpine. Two hours after SE, mice were treated intraperitoneally (i.p.) with androsterone (100-200 mg/kg) or vehicle, and compared with other control groups. Two days after injection, immunohistochemical staining for CB was performed using a hippocampal slice from each mice group. We also used cresyl violet staining to compare changes in hippocampal structures.Two days after pilocarpine-induced SE, androsterone increased the expression of CB in the hippocampus compared with control SE mice. The number of CB-positive cells was 1±0.4 cells/mm(3) in pilocarpine-only group, 14±1.1 cells/mm(3) in pilocarpine plus androsterone 100 mg group and 29±2.5 cells/mm(3) in pilocarpine plus androsterone 200 mg group (p<0.001).These results suggest that the neuroprotective effect of androsterone after pilocarpine- induced SE may be mediated by an increased expression of CB.
- Anticonvulsant effects of acute treatment with cyane-carvone at repeated oral doses in epilepsy models. [JOURNAL ARTICLE]
- Pharmacol Biochem Behav 2014 Jun 23.
Epilepsy affects about 40 million people worldwide. Many drugs block seizures, but have little effect in preventing or curing this disease. So the search for new drugs for epilepsy treatment using animal models prior to testing in humans is important. Increasingly pharmaceutical industries invest in the Research & Drug Development area to seek safe and effective new therapeutic alternatives to the currently available epilepsy treatment. In this perspective, natural compounds have been investigated in epilepsy models, particularly the monoterpenes obtained from medicinal plants. In our study we investigated the effects of cyane-carvone (CC), a synthetic substance prepared from natural a monoterpene, carvone, against pilocarpine- (PILO), pentylenetetrazole- (PTZ) and picrotoxine (PTX)-induced seizures in mice after acute treatment with repeated oral doses (CC 25, 50 and 75 mg/kg) for 14 days. CC in all doses tested showed increase in latency to first seizure, decrease in percentages of seizuring animals as well as reduction percentages of dead animals (p<0.05) in PILO, PTZ and PTX groups when compared with vehicle. However, these effects were not reversed by flumazenil, benzodiazepine (BZD) antagonist used to investigate the CC action mechanism. Our results suggest that acute treatment with CC at the doses tested can exert anticonvulsant effects in PILO, PTZ and PTX epilepsy models. In addition, our data suggest that CC could act in an allosteric site of GABAA, which would be different from the site in which BDZ acts, since flumazenil was not able to reverse any of CC effects on the modulation of seizure parameters related with epilepsy models investigated. New studies should be conducted to investigate CC effects in other neurotransmitter systems. Nevertheless, our study reinforces the hypothesis that CC could be used, after further research, as a new pharmaceutical formulation and a promising alternative for epilepsy treatment, since it showed anticonvulsant effects.
- Identification of Endogenous Reference Genes for the Analysis of microRNA Expression in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy. [JOURNAL ARTICLE]
- PLoS One 2014; 9(6):e100529.
Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.
- Amiloride and SN-6 Suppress Audiogenic Seizure Susceptibility in Genetically Epilepsy-Prone Rats. [JOURNAL ARTICLE]
- CNS Neurosci Ther 2014 Jun 20.
We have recently reported that amiloride, a potent and nonselective blocker of acid-sensing ion channels, prevents the development of pilocarpine-induced seizures and status epilepticus. Amiloride is also known to suppress the activity of Na(+) /Ca(2+) and Na(+) /H(+) exchangers that have been implicated in the pathophysiology of seizures. Here, we evaluated the effects of amiloride, SN-6 (a potent blocker of Na(+) /Ca(2+) exchangers) and zoniporide (a potent blocker of Na(+) /H(+) exchangers) on acoustically evoked seizures (audiogenic seizures, AGS) in genetically epilepsy-prone rats (GEPR-3s), a model of inherited generalized epilepsy.Male, six-week-old GEPR-3s were used. The GEPR-3s were tested for AGS susceptibility before and after treatment with various doses of amiloride, SN-6, and zoniporide (1, 3, 10, and 30 mg/kg; per os).We found that pretreatment with amiloride and SN-6 markedly reduced the incidence and severity of AGS in the GEPR-3s. In contrast, administration of zoniporide only minimally reduced the incidence and severity of AGS in the GEPR-3s. A combination of noneffective doses of SN-6 and zoniporide also suppressed AGS susceptibility in the GEPR-3s.These findings suggest acid-sensing ion channels and the Na(+) /Ca(2+) exchanger may play an important role in the pathophysiology of inherited AGS susceptibility in the GEPR-3s.
- Metrifonate, like acetylcholine, up-regulates neurotrophic activity of cultured rat astrocytes. [Journal Article]
- Pharmacol Rep 2014 Aug; 66(4):618-23.
Metrifonate is an inhibitor of acetylcholinesterase (AChE). Several studies confirmed its positive effects on cognitive impairment in Alzheimer's disease but it was due to adverse events withdrawn from clinical trials. Based on the importance of astrocytes in physiological and pathological brain activities we investigated the impact of metrifonate and, for comparison, acetylcholine on intrinsic neurotrophic activity in these cells.Metabolic activity, intracellular adenosine 5'-triphosphate (ATP) levels and lactate dehydrogenase (LDH) release was measured to examine the impact of metrifonate on viability and integrity of cultured rat cortical astrocytes. The influence of metrifonate, acetylcholine and selective cholinergic ligands on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) synthesis and secretion was determined by specific two-site enzyme immunoassays.Exposure of cultured astrocytes to metrifonate displayed no toxic effects on cell viability. Metrifonate and acetylcholine potently and transiently elevated NGF and BDNF, but not NT-3, protein levels and secretion with different intensity and time frame of their maximal response. Stimulatory effect on NGF was mimicked by selective nicotinic receptor agonist nicotine and completely blocked by nicotinic antagonist mecamylamine. The impact on BDNF synthesis was mimicked by muscarinic receptor agonist pilocarpine and abolished by selective muscarinic antagonist scopolamine.Metrifonate up-regulates astrocytic NGF and BDNF synthesis in the same manner as acetylcholine, their effect depends on different cholinergic pathways. These results suggest a trophic role of metrifonate, based on a well-known neurotrophic activity of NGF and BDNF in vivo.
- Temporally unstructured electrical stimulation to the amygdala suppresses behavioral chronic seizures of the pilocarpine animal model. [JOURNAL ARTICLE]
- Epilepsy Behav 2014 Jun 13.:159-164.
Electrical stimulation applied to the basolateral amygdala in the pentylenetetrazole animal model of seizures may result in either a proconvulsant or an anticonvulsant effect depending on the interpulse intervals used: periodic or nonperiodic, respectively. We tested the effect of this electrical stimulation temporal coding on the spontaneous and recurrent behavioral seizures produced in the chronic phase of the pilocarpine animal model of temporal lobe epilepsy, an experimental protocol that better mimics the human condition. After 45days of the pilocarpine-induced status epilepticus, male Wistar rats were submitted to a surgical procedure for the implantation of a bipolar electrical stimulation electrode in the right basolateral amygdala and were allowed to recover for seven days. The animals were then placed in a glass box, and their behaviors were recorded daily on DVD for 6h for 4 consecutive days (control period). Spontaneous recurrent behavioral seizures when showed in animals were further recorded for an extra 4-day period (treatment period), under periodic or nonperiodic electrical stimulation. The number, duration, and severity of seizures (according to the modified Racine's scale) during treatment were compared with those during the control period. The nonperiodically stimulated group displayed a significantly reduced total number and duration of seizures. There was no difference between control and treatment periods for the periodically stimulated group. Results corroborate previous findings from our group showing that nonperiodic electrical stimulation has a robust anticonvulsant property. In addition, results from the pilocarpine animal model further strengthen nonperiodic electrical stimulation as a valid therapeutic approach in current medical practice. Our working hypothesis is that temporally unstructured electrical stimulation may wield its effect by desynchronizing neural networks involved in the ictogenic process.
- Behavioral and neurochemical studies in mice pretreated with garcinielliptone FC in pilocarpine-induced seizures. [JOURNAL ARTICLE]
- Pharmacol Biochem Behav 2014 Jun 6.
Garcinielliptone FC (GFC) isolated from hexanic fraction seed extract of species Platonia insignis Mart. It is widely used in folk medicine to treat skins diseases in both humans and animals as well as the seed decoction has been used to treat diarrheas and inflammatory diseases. However, there is no research on GFC effects in central nervous system of rodents. The present study aimed to evaluate the GFC effects at doses of 25, 50 or 75mg/kg on seizure parameters to determine their anticonvulsant activity and its effects on amino acids (γ-aminobutyric acid (GABA), glutamine, aspartate and glutathione) levels as well as on acetylcholinesterase (AChE) activity in mice hippocampus after seizures. GFC produced an increased latency to first seizure, at doses 25mg/kg (20.12±2.20min), 50mg/kg (20.95±2.21min) or 75mg/kg (23.43±1,99min) when compared with seized mice. In addition, in GABA content of mice hippocampus treated with GFC75 plus P400 showed an increase of 46.90% when compared seized mice. In aspartate, glutamine and glutamate levels were detected a decrease of 5.21%, 13.55% and 21.80%, respectively in mice hippocampus treated with GFC75 plus P400 when compared with seized mice. Hippocampus mice treated with GFC75 plus P400 showed an increase in AChE activity (63.30%) when compared with seized mice. The results indicate that GFC can exert anticonvulsant activity and reduce the frequency of installation of pilocarpine-induced status epilepticus, as demonstrated by increase in latency to first seizure and decrease in mortality rate of animals. In conclusion, our data suggest that GFC may influence in epileptogenesis and promote anticonvulsant actions in pilocarpine model by modulating the GABA and glutamate contents and of AChE activity in seized mice hippocampus. This compound may be useful to produce neuronal protection and it can be considered as an anticonvulsant agent.