Inherited pain hypersensitivity and increased anxiety-like behaviors are associated with genetic epilepsy in Wistar Audiogenic Rats: Short- and long-term effects of acute and chronic seizures on nociception and anxiety.

Anxiety and pain hypersensitivity are neurobehavioral comorbidities commonly reported by patients with epilepsies, and preclinical models are suitable to investigate the neurobiology of behavioral and neuropathological alterations associated with these epilepsy-related comorbidities. This work aimed to characterize endogenous alterations in nociceptive threshold and anxiety-like behaviors in the Wistar Audiogenic Rat (WAR) model of genetic epilepsy. We also assessed the effects of acute and chronic seizures on anxiety and nociception. WARs from acute and chronic seizure protocols were divided into two groups to assess short- and long-term changes in anxiety (1 day or 15 days after seizures, respectively). To assess anxiety-like behaviors, the laboratory animals were submitted to the open field, light-dark box, and elevated plus maze tests. The von Frey, acetone, and hot plate tests were used to measure the endogenous nociception in seizure-free WARs, and postictal antinociception was recorded at 10, 30, 60, 120, 180 min, and 24 h after seizures. Seizure-free WARs presented increased anxiety-like behaviors and pain hypersensitivity, displaying mechanical and thermal allodynia (to heat and cold stimuli) in comparison to nonepileptic Wistar rats. Potent postictal antinociception that persisted for 120 to 180 min was detected after acute and chronic seizures. Additionally, acute and chronic seizures have magnified the expression of anxiety-like behaviors when assessed at 1 day and 15 days after seizures. Behavioral analysis indicated more severe and persistent anxiogenic-like alterations in WARs submitted to acute seizures. Therefore, WARs presented pain hypersensitivity and increased anxiety-like behaviors endogenously associated with genetic epilepsy. Acute and chronic seizures induced postictal antinociception in response to mechanical and thermal stimuli and increased anxiety-like behaviors when assessed 1 day and 15 days later. These findings support the presence of neurobehavioral alterations in subjects with epilepsy and shed light on the use of genetic models to characterize neuropathological and behavioral alterations associated with epilepsy.

Individuals in Collaborative Governance for Environmental Management.

Analyzing the effect of individual participants on collaborative governance processes in environmental management has been elusive due to lack of theoretical frameworks and data limitations. This study uses pattern matching to contrast identity theory with original data from 7 individuals participating in waste management and urban agriculture collaboration in Florianópolis, Brazil. What started as a self-organized initiative to manage an environmental problem, due to precarious waste management services, was scaled up to a citywide policy. Findings demonstrate that as the collaboration evolved over time, individual participants in municipal government transitioned between roles, organizations, and departments which affected their influence on the collaboration according to two transition styles: integrators (overlapping different roles) and segmenters (aligning roles with contexts without ambiguity). While the integrator-style participants were key to increasing sectoral diversity during the activation stage of the collaboration to produce innovative actions, segmenters contributed to formalizing the collaboration with appropriate institutional designs. However, the success of the collaboration after the institutionalization stage depended on the individual transition style and the power of municipal agents to have agency for influencing the collaboration. These findings have implications for adapting collaborative settings to respond to contextual changes that involve urban environmental issues.

Are Predator Smell (TMT)-Induced Behavioral Alterations in Rats Able to Inhibit Seizures?

We aimed to evaluate the chemical and behavioral effects of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) after olfactory exposure and to verify their influence in the expression of acute audiogenic seizures in the Wistar Audiogenic Rat (WAR) strain. PROTOCOL 1: TMT gas chromatography was applied to define odor saturation in a chamber to different concentrations, time required for saturation and desaturation, and if saturation was homogeneous. Also, male Adult Wistar rats were exposed to saline (SAL) or to different TMT concentrations and their behaviors were evaluated (neuroethology). PROTOCOL 2: Male adult WARs were exposed for 15 s to SAL or TMT, followed by sound stimulation for 1 min or until tonic-clonic convulsion. Behavioral analysis included latencies (wild running and tonic-clonic convulsion), seizure severity indexes, and neuroethology. Gas chromatography established a saturation homogeneous to different concentrations of TMT, indicating that saturation and desaturation occurred in 30 min. TMT triggered fear-like or aversion-like reactions associated with reduction in motor activity and in grooming behavior, in the 2 highest concentrations. Pure TMT presented anticonvulsant properties, such as less-severe seizure phenotype, as well as a decrease in tonic-clonic convulsion expression. TMT elicited fear-like or aversion-like behaviors in Wistar and WAR and can be utilized in a quantifiable and controllable way. Our results suggested possible antagonism between "fear-related" or "aversion-related" and "seizure-related" networks.

Divergent brain changes in two audiogenic rat strains: A voxel-based morphometry and diffusion tensor imaging comparison of the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR).

Acoustically evoked seizures (e.g., audiogenic seizures or AGS) are common in models of inherited epilepsy and occur in a variety of species including rat, mouse, and hamster. Two models that have been particularly well studied are the genetically epilepsy prone rat (GEPR-3) and the Wistar Audiogenic Rat (WAR) strains. Acute and repeated AGS, as well as comorbid conditions, displays a close phenotypic overlap in these models. Whether these similarities arise from convergent or divergent structural changes in the brain remains unknown. Here, we examined the brain structure of Sprague Dawley (SD) and Wistar (WIS) rats, and quantified changes in the GEPR-3 and WAR, respectively. Brains from adult, male rats of each strain (n=8-10 per group) were collected, fixed, and embedded in agar and imaged using a 7 tesla Bruker MRI. Post-acquisition analysis included voxel-based morphometry (VBM), diffusion tensor imaging (DTI), and manual volumetric tracing. In the VBM analysis, GEPR-3 displayed volumetric changes in brainstem structures known to be engaged by AGS (e.g., superior and inferior colliculus, periaqueductal grey) and in forebrain structures (e.g., striatum, septum, nucleus accumbens). WAR displayed volumetric changes in superior colliculus, and a broader set of limbic regions (e.g., hippocampus, amygdala/piriform cortex). The only area of significant overlap in the two strains was the midline cerebellum: both GEPR-3 and WAR showed decreased volume compared to their control strains. In the DTI analysis, GEPR-3 displayed decreased fractional anisotropy (FA) in the corpus callosum, posterior commissure and commissure of the inferior colliculus (IC). WAR displayed increased FA only in the commissure of IC. These data provide a biological basis for further comparative and mechanistic studies in the GEPR-3 and WAR models, as well as provide additional insight into commonalities in the pathways underlying AGS susceptibility and behavioral comorbidity.

Intense olfactory stimulation blocks seizures in an experimental model of epilepsy.

There are reports of patients whose epileptic seizures are prevented by means of olfactory stimulation. Similar findings were described in animal models of epilepsy, such as the electrical kindling of amygdala, where olfactory stimulation with toluene (TOL) suppressed seizures in most rats, even when the stimuli were 20% above the threshold to evoke seizures in already kindled animals. The Wistar Audiogenic Rat (WAR) strain is a model of tonic-clonic seizures induced by acute acoustic stimulation, although it also expresses limbic seizures when repeated acoustic stimulation occurs - a process known as audiogenic kindling (AK). The aim of this study was to evaluate whether or not the olfactory stimulation with TOL would interfere on the behavioral expression of brainstem (acute) and limbic (chronic) seizures in the WAR strain. For this, animals were exposed to TOL or saline (SAL) and subsequently exposed to acoustic stimulation in two conditions that generated: I) acute audiogenic seizures (only one acoustic stimulus, without previous seizure experience before of the odor test) and II) after AK (20 acoustic stimuli [2 daily] before of the protocol test). We observed a decrease in the seizure severity index of animals exposed only to TOL in both conditions, with TOL presented 20s before the acoustic stimulation in both protocols. These findings were confirmed by behavioral sequential analysis (neuroethology), which clearly indicated an exacerbation of clusters of specific behaviors such as exploration and grooming (self-cleaning), as well as significant decrease in the expression of brainstem and limbic seizures in response to TOL. Thus, these data demonstrate that TOL, a strong olfactory stimulus, has anticonvulsant properties, detected by the decrease of acute and AK seizures in WARs.

Morphofunctional alterations in the olivocochlear efferent system of the genetic audiogenic seizure-prone hamster GASH:Sal.

The genetic audiogenic seizure hamster (GASH:Sal) is a model of a form of reflex epilepsy that is manifested as generalized tonic-clonic seizures induced by external acoustic stimulation. The morphofunctional alterations in the auditory system of the GASH:Sal that may contribute to seizure susceptibility have not been thoroughly determined. In this study, we analyzed the olivocochlear efferent system of the GASH:Sal from the organ of Corti, including outer and inner hair cells, to the olivocochlear neurons, including shell, lateral, and medial olivocochlear (LOC and MOC) neurons that innervate the cochlear receptor. To achieve this, we carried out a multi-technical approach that combined auditory hearing screenings, scanning electron microscopy, morphometric analysis of labeled LOC and MOC neurons after unilateral Fluoro-Gold injections into the cochlea, and 3D reconstruction of the lateral superior olive (LSO). Our results showed that the GASH:Sal exhibited higher auditory brain response (ABR) thresholds than their controls, as well as absence of distortion-product of otoacoustic emissions (DPOAEs) in a wide range of frequencies. The ABR and DPOAE results also showed differences between the left and right ears, indicating asymmetrical hearing alterations in the GASH:Sal. These alterations in the peripheral auditory activity correlated with morphological alterations. At the cochlear level, the scanning electron microscopy analysis showed marked distortions of the stereocilia from basal to apical cochlear turns in the GASH:Sal, which were not observed in the control hamsters. At the brainstem level, MOC, LOC, and shell neurons had reduced soma areas compared with control animals. This LOC neuron shrinkage contributed to reduction in the LSO volume of the GASH:Sal as shown in the 3D reconstruction analysis. Our study demonstrated that the morphofunctional alterations of the olivocochlear efferent system are innate components of the GASH:Sal, which might contribute to their susceptibility to audiogenic seizures. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Looking for complexity in quantitative semiology of frontal and temporal lobe seizures using neuroethology and graph theory.

Epileptic syndromes and seizures are the expression of complex brain systems. Because no analysis of complexity has been applied to epileptic seizure semiology, our goal was to apply neuroethology and graph analysis to the study of the complexity of behavioral manifestations of epileptic seizures in human frontal lobe epilepsy (FLE) and temporal lobe epilepsy (TLE). We analyzed the video recordings of 120 seizures of 18 patients with FLE and 28 seizures of 28 patients with TLE. All patients were seizure-free >1 year after surgery (Engel Class I). All patients' behavioral sequences were analyzed by means of a glossary containing all behaviors and analyzed for neuroethology (Ethomatic software). The same series were used for graph analysis (CYTOSCAPE). Behaviors, displayed as nodes, were connected by edges to other nodes according to their temporal sequence of appearance. Using neuroethology analysis, we confirmed data in the literature such as in FLE: brief/frequent seizures, complex motor behaviors, head and eye version, unilateral/bilateral tonic posturing, speech arrest, vocalization, and rapid postictal recovery and in the case of TLE: presence of epigastric aura, lateralized dystonias, impairment of consciousness/speech during ictal and postictal periods, and development of secondary generalization. Using graph analysis metrics of FLE and TLE confirmed data from flowcharts. However, because of the algorithms we used, they highlighted more powerfully the connectivity and complex associations among behaviors in a quite selective manner, depending on the origin of the seizures. The algorithms we used are commonly employed to track brain connectivity from EEG and MRI sources, which makes our study very promising for future studies of complexity in this field.

Gentamicin conditioning confers auditory protection against noise trauma.

Auditory conditioning consists of the pre-exposure to low levels of a potential harmful agent to protect against a subsequent harmful presentation. The agent that was first tested was noise. This paradigm was more recently successfully tested with other agents. Nonetheless, the vast majority of the studies utilize the same agent to condition and to cause the trauma. The aim of this study was to verify whether conditioning with an agent different from the agent used to cause the trauma can also be effective. Thus, the following groups were organized: group Cont, which is the noise trauma control group, was exposed to 110-dB broadband noise centered at 4 kHz for 72 h; group Gent, which is the gentamicin conditioning control group, was administered 30 mg/kg of gentamicin daily for 30 consecutive days; and group Expt was conditioned with gentamicin similarly to group Gent and then subjected to a noise trauma similarly to group Cont. The animals were functionally and morphologically evaluated through the measurement of the auditory brainstem response and scanning electron microscopy, respectively. The following variables were investigated: outer hair cell injury and auditory threshold shift. The group that was conditioned with the drug exhibited significantly less outer hair cell damage, 10.8 and 22.9%, respectively (p = 0.0146), although did not maintain the proper functioning of the auditory system. We, therefore, conclude that conditioning with a different agent from that used to cause the trauma is effective, which suggests that both agents that were used promote similar mechanisms of self-protection.

Audiogenic Seizures in the Streptozotocin-Induced Rat Alzheimer's Disease Model.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative and progressive disorder with no cure and constant failures in clinical trials. The main AD hallmarks are amyloid-ß (Aß) plaques, neurofibrillary tangles, and neurodegeneration. However, many other events have been implicated in AD pathogenesis. Epilepsy is a common comorbidity of AD and there is important evidence indicating a bidirectional link between these two disorders. Some studies suggest that disturbed insulin signaling might play an important role in this connection. OBJECTIVE: To understand the effects of neuronal insulin resistance in the AD-epilepsy link. METHODS: We submitted the streptozotocin (STZ) induced rat AD Model (icv-STZ AD) to an acute acoustic stimulus (AS), a known trigger of seizures. We also assessed animals' performance in the memory test, the Morris water maze and the neuronal activity (c-Fos protein) induced by a single audiogenic seizure in regions that express high levels of insulin receptors. RESULTS: We identified significant memory impairment and seizures in 71.43% of all icv-STZ/AS rats, in contrast to 22.22% of the vehicle group. After seizures, icv-STZ/AS rats presented higher number of c-Fos immunopositive cells in hippocampal, cortical, and hypothalamic regions. CONCLUSION: STZ may facilitate seizure generation and propagation by impairment of neuronal function, especially in regions that express high levels of insulin receptors. The data presented here indicate that the icv-STZ AD model might have implications not only for AD, but also for epilepsy. Finally, impaired insulin signaling might be one of the mechanisms by which AD presents a bidirectional connection to epilepsy.

Absence-like Seizures, Cortical Oscillations Abnormalities and Decreased Anxiety-like Behavior in Wistar Audiogenic Rats with Cortical Microgyria.

Wistar Audiogenic Rats (WAR) is an inbred rodent strain susceptible to acute auditory stimulation-induced seizures. However, spontaneous epileptic seizures (SES) and their associated electroencephalogram (EEG) abnormalities have not been reported in WAR kindled animals. The same is true for naïve WARs (without sound-induced seizures). An approach to increment epileptogenesis and SES is to use a second insult to be added to the genetic background. Here, we used adult naïve WARs with microgyria induced by neonatal cortical freeze-lesion (FL) to evaluate the occurrence of SES and the modification in cortical oscillation patterns and behavior. The neonatal cortical FL was performed in Wistar and naïve WARs (Wis-FL and WAR-FL). Sham animals were used as controls (Wistar-S and WAR-S). Video-EEG recordings and behavioral tasks were performed during adulthood. Surprisingly, spike-waive discharges (SWD) events associated with behavior arrest were detected in WAR-S rats. Those events increased in duration and number in WAR-FL animals. The EEG quantitative analysis showed decreased power of cortical delta, theta and beta oscillations in WAR-S, decreased power of cortical fast gamma (FG) oscillations in WARs, independent of microgyria, and decreased interhemispheric synchrony for delta and FG with stronger coupling in delta and theta-FG oscillations in FL animals. The WARs, regardless of microgyria, had reduced locomotor activity, but only WAR-FL animals had reduced anxiety-like behavior. Microgyria in naïve WARs intensified SWD events associated with behavior arrest that could reflect absence-like seizures and abnormal cortical oscillations, and reduced anxiety-like behavior indicating that WAR-FL could be a reliable model to study epileptogenesis.

A Genetic Model of Epilepsy with a Partial Alzheimer's Disease-Like Phenotype and Central Insulin Resistance.

Studies have suggested an important connection between epilepsy and Alzheimer's disease (AD), mostly due to the high number of patients diagnosed with AD who develop epileptic seizures later on. However, this link is not well understood. Previous studies from our group have identified memory impairment and metabolic abnormalities in the Wistar audiogenic rat (WAR) strain, a genetic model of epilepsy. Our goal was to investigate AD behavioral and molecular alterations, including brain insulin resistance, in naïve (seizure-free) animals of the WAR strain. We used the Morris water maze (MWM) test to evaluate spatial learning and memory performance and hippocampal tissue to verify possible molecular and immunohistochemical alterations. WARs presented worse performance in the MWM test (p < 0.0001), higher levels of hyperphosphorylated tau (S396) (p < 0.0001) and phosphorylated glycogen synthase kinase 3 (S21/9) (p < 0.05), and lower insulin receptor levels (p < 0.05). Conversely, WARs and Wistar controls present progressive increase in amyloid fibrils (p < 0.0001) and low levels of soluble amyloid-ß. Interestingly, the detected alterations were age-dependent, reaching larger differences in aged than in young adult animals. In summary, the present study provides evidence of a partial AD-like phenotype, including altered regulation of insulin signaling, in a genetic model of epilepsy. Together, these data contribute to the understanding of the connection between epilepsy and AD as comorbidities. Moreover, since both tau hyperphosphorylation and altered insulin signaling have already been reported in epilepsy and AD, these two events should be considered as important components in the interconnection between epilepsy and AD pathogenesis and, therefore, potential therapeutic targets in this field.

Behavioral and EEG effects of GABAergic manipulation of the nigrotectal pathway in the Wistar audiogenic rat strain.

The superior colliculus (SC), substantia nigra pars reticulata (SNPr), and striatum have been characterized as important structures involved in the modulation of seizure activity. In the current study, bicuculline (GABA(A) antagonist) and muscimol (GABA(A) agonist) were microinjected into the deep layers of either the anterior SC (aSC) or posterior SC (pSC) of genetically developed Wistar audiogenic rats. Behavior and EEG activity were studied simultaneously. Only muscimol microinjected into the pSC had behavioral and EEG anticonvulsant effects in Wistar audiogenic rats, eliciting EEG oscillation changes in both SNPr and pSC, primarily during tonic seizures. The SC of Wistar audiogenic rats thus comprises two functionally different subregions, pSC and aSC, defined by distinct behavioral and EEG features. The pSC has proconvulsant audiogenic seizure activity in Wistar audiogenic rats. Our data suggest that this phenomenon may be a consequence of the genetic selection of the Wistar audiogenic rat strain.

Neuroendocrine changes in the hypothalamic-neurohypophysial system in the Wistar audiogenic rat (WAR) strain submitted to audiogenic kindling.

The Wistar audiogenic rat (WAR) strain is used as an animal model of epilepsy, which when submitted to acute acoustic stimulus presents tonic-clonic seizures, mainly dependent on brainstem (mesencephalic) structures. However, when WARs are exposed to chronic acoustic stimuli (audiogenic kindling-AK), they usually present tonic-clonic seizures, followed by limbic seizures, after recruitment of forebrain structures such as the cortex, hippocampus and amygdala. Although some studies have reported that hypothalamic-hypophysis function is also altered in WAR through modulating vasopressin (AVP) and oxytocin (OXT) secretion, the role of these neuropeptides in epilepsy still is controversial. We analyzed the impact of AK and consequent activation of mesencephalic neurocircuits and the recruitment of forebrain limbic (LiR) sites on the hypothalamic-neurohypophysial system and expression of Avpr1a and Oxtr in these structures. At the end of the AK protocol, nine out of 18 WARs presented LiR. Increases in both plasma vasopressin and oxytocin levels were observed in WAR when compared to Wistar rats. These results were correlated with an increase in the expressions of heteronuclear (hn) and messenger (m) RNA for Oxt in the paraventricular nucleus (PVN) in WARs submitted to AK that presented LiR. In the paraventricular nucleus, the hnAvp and mAvp expressions increased in WARs with and without LiR, respectively. There were no significant differences in Avp and Oxt expression in supraoptic nuclei (SON). Also, there was a reduction in the Avpr1a expression in the central nucleus of the amygdala and frontal lobe in the WAR strain. In the inferior colliculus, Avpr1a expression was lower in WARs after AK, especially those without LiR. Our results indicate that both AK and LiR in WARs lead to changes in the hypothalamic-neurohypophysial system and its receptors, providing a new molecular basis to better understaind epilepsy.

Putative Causal Variant on Vlgr1 for the Epileptic Phenotype in the Model Wistar Audiogenic Rat.

Wistar Audiogenic Rat is an epilepsy model whose animals are predisposed to develop seizures induced by acoustic stimulation. This model was developed by selective reproduction and presents a consistent genetic profile due to the several generations of inbreeding. In this study, we performed an analysis of WAR RNA-Seq data, aiming identified at genetic variants that may be involved in the epileptic phenotype. Seventeen thousand eighty-five predicted variants were identified as unique to the WAR model, of which 15,915 variants are SNPs and 1,170 INDELs. We filter the predicted variants by pre-established criteria and selected five for validation by Sanger sequencing. The genetic variant c.14198T>C in the Vlgr1 gene was confirmed in the WAR model. Vlgr1 encodes an adhesion receptor that is involved in the myelination process, in the development of stereocilia of the inner ear, and was already associated with the audiogenic seizures presented by the mice Frings. The transcriptional quantification of Vlgr1 revealed the downregulation this gene in the corpus quadrigeminum of WAR, and the protein modeling predicted that the mutated residue alters the structure of a domain of the VLGR1 receptor. We believe that Vlgr1 gene may be related to the predisposition of WAR to seizures and suggest the mutation Vlgr1/Q4695R as putative causal variant, and the first molecular marker of the WAR strain.

The neurobiological substrates of behavioral manifestations during temporal lobe seizures: a neuroethological and ictal SPECT correlation study.

Ictal behavior coupled with SPECT findings during 28 seizures in patients with temporal lobe epilepsy (TLE) with unilateral hippocampal sclerosis (13 left; 15 right) was displayed as flowcharts from right-sided (RTLE) plus left-sided (LTLE) seizures. Ictal SPECT was classified blind to neuroethology. Behaviors were categorized as ipsilateral to the epileptogenic zone (IL), contralateral to the epileptogenic zone (CL), or bilateral. SPECT intensity and region were categorized as IL or CL to the epileptogenic zone. All patients developed automatisms and had hyperperfusion in their temporal lobes. Patients' verbal responses to questions had statistical interactions in RTLE but not in LTLE sum. Most CL dystonic posturing was correlated to IL basal ganglia hyperperfusion. Basal ganglia activation occurred in seizures without dystonic posturing and CL manual automatisms, and lack of IL dystonic posturing and the presence of CL cerebellar hemispheric hyperperfusion were also observed. Coupling of neuroethology and SPECT findings reliably evaluates ictal behavior and functionality of associated brain areas.

Alterations in brainstem auditory processing, the acoustic startle response and sensorimotor gating of startle in Wistar audiogenic rats (WAR), an animal model of reflex epilepsies.

While acute audiogenic seizures in response to acoustic stimulus appear as an alteration in sensory-motor processing in the brainstem, the repetition of the stimulus leads to the spread of epileptic activity to limbic structures. Here, we investigated whether animals of the Wistar Audiogenic Rat (WAR) strain, genetically selected by inbreeding for seizure susceptibility, would have alterations in their auditory response, assessed by the auditory brainstem responses (ABR) and sensory-motor gating, measured as pre-pulse inhibition (PPI), which could be related to their audiogenic seizures susceptibility or severity. We did not find differences between the amplitudes and latencies of ABR waves in response to clicks for WARs when compared to Wistars. Auditory gain and symmetry between ears were also similar. However, hearing thresholds in response to some tones were lower and amplitudes of wave II were larger in WARs. WARs had smaller acoustic startle reflex amplitudes and the percentages of startle inhibited by an acoustic prepulse were higher for WARs than for Wistars. However, no correlation was found between these alterations and brainstem-dependent seizure severity or limbic seizure frequency during audiogenic kindling. Our data show that while WARs present moderate alterations in primary auditory processing, the sensory motor gating measured in startle/PPI tests appears to be more drastically altered. The observed changes might be correlated with audiogenic seizure susceptibility but not seizures severity.

Inflammatory markers in the hippocampus after audiogenic kindling.

Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the audiogenic kindling protocol with a loud broad-band noise. We measured pro and anti-inflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure severity, we did not observe differences in the pro inflammatory cytokines IL-1ß, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate.

Energy Metabolism and Redox State in Brains of Wistar Audiogenic Rats, a Genetic Model of Epilepsy.

The Wistar Audiogenic Rat (WAR) strain is a genetic model of epilepsy, specifically brainstem-dependent tonic-clonic seizures, triggered by acute auditory stimulation. Chronic audiogenic seizures (audiogenic kindling) mimic temporal lobe epilepsy, with significant participation of the hippocampus, amygdala, and cortex. The objective of the present study was to characterize the mitochondrial energy metabolism in hippocampus and cortex of WAR and verify its relationship with seizure severity. Hippocampus of WAR naïve (no seizures) presented higher oxygen consumption in respiratory states related to the maximum capacities of phosphorylation and electron transfer system, elevated mitochondrial density, lower GSH/GSSG and catalase activity, and higher protein carbonyl and lactate contents, compared with their Wistar counterparts. Audiogenic kindling had no adding functional effect in WAR, but in Wistar, it induced the same alterations observed in the audiogenic strain. In the cortex, WAR naïve presented elevated mitochondrial density, lower GSH/GSSG and catalase activity, and higher protein carbonyl levels. Chronic acoustic stimulation in Wistar induced the same alterations in cortex and hippocampus. Mainly in the hippocampus, WAR naïve presented elevated mRNA expression of glucose, lactate and excitatory amino acids transporters, several glycolytic enzymes, lactate dehydrogenase, and Na+/K+ ATPase in neurons and in astrocytes. In vivo treatment with mitochondrial uncoupler 2,4-dinitrophenol (DNP) or N-acetylcysteine (NAC) in WAR had no effect on mitochondrial metabolism, but lowered oxidative stress. Unlike DNP, NAC downregulated all enzyme genes involved in glucose and lactate uptake, and metabolism in neurons and astrocytes. Additionally, it was able to reduce brainstem seizure severity in WAR. In conclusion, in WAR naïve animals, both cerebral cortex and hippocampus display elevated mitochondrial density and/or activity associated with oxidative damage, glucose and lactate metabolism pathways upregulation, and increased Na+/K+ ATPase mRNA expression. Only in vivo treatment with NAC was able to reduce seizure severity of kindled WARs, possibly via down regulation of glucose/lactate metabolism. Taken together, our results are a clear contribution to the field of mitochondrial metabolism associated to epileptic seizures.

Differences in the expression pattern of P-glycoprotein and MRP1 in low-grade and high-grade gliomas.

Multidrug resistance in gliomas is the major challenges in the clinical setting. We investigated the expression of P-glycoprotein (Pgp) and multidrug resistance-related protein 1 (MRP1) in 50 gliomas using immunohistochemistry. Compared to Pgp, MRP1 positivity was observed in highest percentage of gliomas grade IV samples (p = 0.008). Unlike MRP1 expression observed in high-grade, gliomas grade II exhibited a greater number of Pgp positive samples as compared to grades III and IV (p = 0.026). Our results suggest that the difference between the histological grade gliomas regarding MRP1 and Pgp expression must have implications in the choice of chemotherapeutic protocols.

Ototoxicity and otoprotection in the inner ear of guinea pigs using gentamicin and amikacin: ultrastructural and functional aspects.

UNLABELLED: Ototoxicity is still a challenge to medicine. The discovery of self-protecting endogenous mechanisms of the outer hair cells associated with their functional and ultra-structural assessment methods has opened new horizons in the understanding and controlling of these mechanisms. AIM: this paper aimed at establishing whether or not underdoses of gentamicin could protect the inner ear against the harmful effects of amikacin, based on these protection mechanisms and determine if the otoacoustic emission amplitudes could be associated with the level of hair cell integrity. MATERIALS AND METHODS: Experimental study. We used 31 guinea pigs. They were injected with saline solution, gentamicin and amikacin, alone and in combinations -intramuscular injections - during 12, 30 and 42 days. The otoacoustic emissions were recorded in the beginning and at the end of the experiment, comparing it with the cochlear integrity study carried out by electron microscopy. RESULTS: gentamicin underdoses did not protect the inner ear against amikacin toxicity; the reduction in otoacoustic emissions was strongly associated with an increase in hair cell lesions. CONCLUSION: these findings help understand inner ear otoprotection and ototoxicity. Establishing the correlation between the emissions amplitude an cell integrity plays an important role in the follow up of hair cell damage, with possible monitoring of ototoxicity caused by drugs in humans.