Aquaporin 4 inhibition decreased synthesis of cytokines by acetazolamide in the hippocampus of rats with pentrazol-induced chronic epilepsy.

Epilepsy refers to a clinical syndrome generated by spontaneous seizures in the central nervous system. Epilepsy triggers a complex pathological process including inflammatory response and aquaporin 4 (AQP4) increase. It has been reported that AQP4 helps to enhance the immunological function of the central nervous system in pathological conditions, but the relationship between AQP4 and inflammatory cytokines is poorly understood in chronic epilepsy processes. As an inhibitor of sulfonamide carbonic anhydrase (CA), acetazolamide (AZA) may inhibit water infiltration through AQP4. In this context, pentylenetetrazole (PTZ) is used to induce the chronic epilepsy model in rats to study the chronic epilepsy effects of AQP4 inhibition on proinflammatory cytokine expression in the hippocampus and proinflammatory cytokine quantification analysis of the plasma. Based on the assumption that AQP4 regulates proinflammatory cytokine expression, this article aims to demonstrate this effect in chronic epilepsy of rats. Rats were divided into four groups and were treated with different drugs: saline (Control), acetazolamide (AZA), pentylenetetrazole (PTZ), and pentylenetetrazole plus acetazolamide (PTZ+AZA). The data showed that seizures increased proinflammatory cytokine expression and that AZA significantly inhibited AQP4 expression. Overall, the results suggested that AQP4 inhibition could weaken excitotoxicity in epileptogenesis by reducing proinflammatory cytokines in the hippocampus. The findings provide a new insight into the involvement of cerebral edema insult and proinflammatory cytokines in the process of chronic epilepsy.

Inhibition of long-term potentiation in the schaffer-CA1 pathway by repetitive high-intensity sound stimulation.

High-intensity sound can induce seizures in susceptible animals. After repeated acoustic stimuli changes in behavioural seizure repertoire and epileptic EEG activity might be seen in recruited limbic and forebrain structures, a phenomenon known as audiogenic kindling. It is postulated that audiogenic kindling can produce synaptic plasticity events leading to the spread of epileptogenic activity to the limbic system. In order to test this hypothesis, we investigated if long-term potentiation (LTP) of hippocampal Schaffer-CA1 synapses and spatial navigation memory are altered by a repeated high-intensity sound stimulation (HISS) protocol, consisting of one-minute 120 dB broadband noise applied twice a day for 10 days, in normal Wistar rats and in audiogenic seizure-prone rats (Wistar Audiogenic Rats - WARs). After HISS all WARs exhibited midbrain seizures and 50% of these animals developed limbic recruitment, while only 26% of Wistar rats presented midbrain seizures and none of them had limbic recruitment. In naïve animals, LTP in hippocampal CA1 neurons was induced by 50- or 100-Hz high-frequency stimulation of Schaffer fibres in slices from both Wistar and WAR animals similarly. Surprisingly, HISS suppressed LTP in CA1 neurons in slices from Wistar rats that did not present any seizure, and inhibited LTP in slices from Wistar rats with only midbrain seizures. However HISS had no effect on LTP in CA1 neurons from slices of WARs. Interestingly HISS did not alter spatial navigation and memory in both strains. These findings show that repeated high-intensity sound stimulation prevent LTP of Schaffer-CA1 synapses from Wistar rats, without affecting spatial memory. This effect was not seen in hippocampi from audiogenic seizure-prone WARs. In WARs the link between auditory stimulation and hippocampal LTP seems to be disrupted which could be relevant for the susceptibility to seizures in this strain.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling.

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1⁻/⁻ mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs.

Limbic epileptogenicity, cell loss and axonal reorganization induced by audiogenic and amygdala kindling in wistar audiogenic rats (WAR strain).

Audiogenic seizures are a model of generalized tonic-clonic brainstem-generated seizures. Repeated induction of audiogenic seizures, in audiogenic kindling (AuK) protocols, generates limbic epileptogenic activity. The present work evaluated associations between permanence of AuK-induced limbic epileptogenicity and changes in cell number/gluzinergic terminal reorganization in limbic structures in Wistar audiogenic rats (WARs). Additionally, we evaluated histological changes after only amygdala kindling (AmK) and only AuK, and longevity of permanence of AuK-induced limbic epileptogenicity, up to 160 days. WARs and Wistar non-susceptible rats were submitted to AuK (80 stimuli) followed by both 50 days without acoustic stimulation and AmK (16 stimuli), only AmK and only AuK. Cell counting and gluzinergic terminal reorganization were assessed, respectively, by using Nissl and neo-Timm histochemistries, 24 h after the last AmK stimulus. Evaluation of behavioral response to a single acoustic stimulus after AuK and up to 160 days without acoustic stimulation was done in another group. AuK-induced limbic epileptogenicity developed in parallel with a decrease in brainstem-type seizure severity during AuK. AmK was facilitated after AuK. Permanence of AuK-induced limbic epileptogenicity was associated with cell loss only in the rostral lateral nucleus of amygdala. Roughly 20 generalized limbic seizures induced by AuK were neither associated with hippocampal cell loss nor mossy fiber sprouting (MFS). AmK developed with cell loss in hippocampal and amygdala nuclei but not MFS. Main changes of gluzinergic terminals after kindling protocols were observed in amygdala, perirhinal and piriform cortices. AuK and AuK-AmK induced a similar number and type of seizures, higher than in AmK. AmK and AuK-AmK were associated with broader cell loss than AuK. Data indicate that permanent AuK-induced limbic epileptogenicity is mainly associated to gluzinergic terminal reorganization in amygdala but not in the hippocampus and with no hippocampal cell loss. Few AmK-induced seizures are associated to broader and higher cell loss than a higher number of AuK-induced seizures.

Presence of claudins mRNA in the brain. Selective modulation of expression by kindling epilepsy.

In the central nervous system, the junctional types that establish and maintain tissue architecture include gap junctions, for cytoplasmic connectivity, and tight junctions, for paracellular and/or cell polarity barriers. Connexins are the integral membrane proteins of gap junctions, whereas occludin and members of the multigene family of claudins form tight junctions. In the brain, there are no transendothelial pathways, as continuous tight junctions are present between the endothelial cells. Thus, they provide a continuous cellular barrier between the blood and the insterstitial fluid. However, several brain pathologies, including epilepsy, are known to alter the permeability of the blood-brain barrier and to cause edema. Therefore, since claudins, as constitutive proteins of tight junctions are likely candidates for modulation under pathological states, we explored their normal pattern of expression in the brain and its modulation by seizures. We found that several members of this family are normally expressed in the hippocampus and cortex. Interestingly, claudin-7 is expressed in the hippocampus but not in the cortex. On the other hand, the expression of claudin-8 is selectively down-regulated in the hippocampus as kindling evolves. These results link for the first time the modulation of expression of a tight junction protein to abnormal neuronal synchronization that could probably be reflected in permeability changes of the blood-brain barrier or edema.

Vesicular GABA transporter mRNA expression in the dentate gyrus and in mossy fiber synaptosomes.

In the normal granule cells of the dentate gyrus, glutamate and both gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) coexist. GAD expression is increased after seizures, and simultaneous glutamatergic and GABAergic neurotransmission from the mossy fibers to CA3 appears, supporting the hypothesis that GABA can be released from the mossy fibers. To sustain GABAergic neurotransmission, the amino acid must be transported into synaptic vesicles. To address this, using RT-PCR we looked for the presence and regulation of expression of the vesicular GABA transporter (VGAT) mRNA in the dentate gyrus and in mossy fiber synaptosomes of control and kindled rats. We found trace amounts of VGAT mRNA in the dentate gyrus and mossy fiber synaptosomes of control rats. In the dentate gyrus of kindled rats with several seizures and of control rats subject to one acute seizure, no changes were apparent either 1 or 24 h after the seizures. However, repetitive synaptic or antidromic activation of the granule cells in slices of control rats in vitro induces an activity-dependent enhancement of VGAT mRNA expression in the dentate. Surprisingly, in the mossy fiber synaptosomes of seizing rats, the levels of VGAT mRNA were significantly higher than in controls. These data show that the granule cells and their mossy fibers, besides containing machinery for the synthesis of GABA, also contain the elements that support its vesiculation. This further supports the notion that local synaptic molecular changes enable mossy fibers to release GABA in response to enhanced excitability.

Antisense oligonucleotides to C-fos reduce postictal seizure susceptibility following fully kindled seizures in rats.

In a previous study we demonstrated that increased FOS expression in the amygdala induced by partial kindling seizures could be attenuated by administering c-Fos specific antisense oligonucleotides. In addition, we found that the administration of c-Fos antisense oligonucleotides at the beginning of the amygdala kindling process facilitated the appearance of stage V kindled seizures. In the present study, we evaluated the effect of the suppression of FOS on the expression and severity of the generalized fully kindled seizures as well as on the susceptibility to additional ictal events during the postictal and interictal periods. We observed that the administration of c-Fos antisense oligonucleotides did not modify the behavioral and electrographic manifestations of generalized stage V kindled seizures. However, c-Fos antisense oligonucleotides significantly reduced the susceptibility to additional ictal events during the postictal refractory period. Hence, the increased FOS protein induced by generalized tonic clonic seizures may participate in postictal mechanisms.