The latency to awake from induced-obstructive sleep apnea is reduced in rats with chronic epilepsy.

OSA is known to increase the risk for SUDEP in persons with epilepsy, but the relationship between these two factors is not clear. Also, there is no study showing the acute responses to obstructive apnea in a chronic epilepsy model. Therefore, this study aimed to characterize cardiorespiratory responses to obstructive apnea and chemoreceptor stimulation in rats. In addition, we analyzed respiratory centers in the brain stem by immunohistochemistry. Epilepsy was induced with pilocarpine. About 30-60 days after the first spontaneous seizure, tracheal and thoracic balloons, and electrodes for recording the electroencephalogram, electromyogram, and electrocardiogram were implanted. Intermittent apneas were made by inflation of the tracheal balloon during wakefulness, NREM sleep, and REM sleep. During apnea, respiratory effort increased, and heart rate fell, especially with apneas made during wakefulness, both in control rats and rats with epilepsy. Latency to awake from apnea was longer with apneas made during REM than NREM, but rats with epilepsy awoke more rapidly than controls with apneas made during REM sleep. Rats with epilepsy also had less REM sleep. Cardiorespiratory responses to stimulation of carotid chemoreceptors with cyanide were similar in rats with epilepsy and controls. Immunohistochemical analysis of Phox2b, tryptophan hydroxylase, and NK1 in brain stem nuclei involved in breathing and sleep (retrotrapezoid nucleus, pre-Bötzinger complex, Bötzinger complex, and caudal raphe nuclei) revealed no differences between control rats and rats with epilepsy. In conclusion, our study showed that rats with epilepsy had a decrease in the latency to awaken from apneas during REM sleep, which may be related to neuroplasticity in some other brain regions related to respiratory control, awakening mechanisms, and autonomic modulation.

Alterations in aortic vasorelaxation in rats with epilepsy induced by the electrical amygdala kindling model.

Cardiovascular alterations are frequently related to epilepsy in both clinical and experimental models, and have been hypothesized as a potential contributor to sudden unexpected death in epilepsy (SUDEP). Further, the frequency of generalized tonic-clonic seizures (GTCS) is a primary risk factor for SUDEP. Therefore, we aimed to evaluate the vascular response of rats subjected to the electrical amygdala kindling model of epilepsy. Male Wistar rats were randomly distributed into the following groups: without seizures (sham, n = 8), 5 GTCS (5 S, n = 5), and 10 GTCS (10 S, n = 6). One day after the last seizure, the rats were euthanized, and the thoracic aorta rings with (E+) and without (E-) endothelium were used to evaluate vascular reactivity ex vivo using the organ bath system. The maximum response to acetylcholine-induced vasorelaxation in the E+ aortic ring was lower in the 5 S group than in the sham and 10 S groups. A reduced concentration of sodium nitroprusside was required to induce vasorelaxation in the E- aortic rings. These results suggest an impairment in endothelial function and alterations in the nitric oxide (NO) pathway. In conclusion, epilepsy altered the vasorelaxation of the aortic rings and the number of seizures influenced these alterations; therefore, an analysis of endothelial function in patients with a high risk of SUDEP may be beneficial.

The number and periodicity of seizures induce cardiac remodeling and changes in micro-RNA expression in rats submitted to electric amygdala kindling model of epilepsy.

Generalized tonic-clonic seizures (GTCS) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). Also, among the several mechanisms underlying SUDEP there is the cardiac dysfunction. So, we aimed to evaluate the impact of the number of seizures on heart function and morphology in rats with epilepsy. Rats were randomized into three groups: Sham (without epilepsy), 5 S, and 10 S groups, referred as rats with epilepsy with a total of 5 or 10 GTCS, respectively. Epilepsy was induced by electrical amygdala kindling. The ventricular function was analyzed by the Langendorff technique and challenged by ischemia/reperfusion protocol. Cardiac fibrosis and hypertrophy were analyzed by histology. We also analyzed cardiac metalloproteinases (MMP2 and MMP9), ERK 1/2 and phosphorylated ERK1/2 (P-ERK) by western blot; microRNA-21 and -320 by RT-PCR; and oxidative stress (TBARS, catalase activity and nitrite) by biochemical analysis. Only the 5S group presented decreased values of ventricular function at before ischemia/reperfusion (baseline): intraventricular systolic pressure, developed intraventricular pressure, positive and negative dP/dt. During ischemia/reperfusion protocol, the variation of the ventricular function did not differ among groups. Both 5S and 10S groups had increased cardiomyocyte hypertrophy and fibrosis compared to Sham, but in the 5S group, these alterations were higher than in the 10S group. The 5S group increased in microRNA-21 and decreased in microRNA-320 expression compared to Sham and the 10S group. The 10S group increased in MMP9 and decreased in P-ERK/ERK expression, and increased in nitrite content compared to both Sham and the 5S group. Therefore, seizures impair cardiac function and morphology, probably through microRNA modulation. The continuation of seizures seems to exert a preconditioning-like stimulus that fails to compensate the cardiac tissue alteration.

Antiepileptic effects of long-term intracerebroventricular infusion of angiotensin-(1-7) in an animal model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy and is often refractory to pharmacological treatment. In this scenario, extensive research has identified components of the renin-angiotensin system (RAS) as potential therapeutic targets. Therefore, the aim of the present study was to evaluate the effects of long-term treatment with angiotensin-(1-7) [Ang-(1-7)] in male Wistar rats with TLE induced by pilocarpine (PILO). Rats with TLE were submitted to intracerebroventricular (icv) infusion of Ang-(1-7) (200 ng/kg/h) for 28 days, starting at the first spontaneous motor seizure (SMS). Body weight, food intake, and SMS were evaluated daily. Behavioral tests and hippocampal protein levels were also evaluated at the end of the treatment. Ang-(1-7) treatment reduced the frequency of SMS and attenuated low anxiety levels, increased locomotion/exploration, and reduced body weight gain that was induced by TLE. Moreover, Ang-(1-7) positively regulated the hippocampal levels of antioxidant protein catalase and antiapoptotic protein B-cell lymphoma 2 (Bcl-2), as well as mammalian target of rapamycin (mTOR) phosphorylation, which were reduced by TLE. The hippocampal up-regulation of angiotensin type 1 receptor induced by TLE was also attenuated by Ang-(1-7), while the Mas receptor (MasR) was down-regulated compared with epilepsy. These data show that Ang-(1-7) presents an antiepileptic effect, increasing neuroprotection markers and reducing SMS frequency, body weight, and behavior impairments found in TLE. Therefore, Ang-(1-7) is a promising coadjutant therapeutic option for the treatment of TLE.

Corrigendum: The Blood-Brain Barrier Breakdown During Acute Phase of the Pilocarpine Model of Epilepsy Is Dynamic and Time-Dependent.

[This corrects the article DOI: 10.3389/fneur.2019.00382.].

The Blood-Brain Barrier Breakdown During Acute Phase of the Pilocarpine Model of Epilepsy Is Dynamic and Time-Dependent.

The maintenance of blood-brain barrier (BBB) integrity is essential for providing a suitable environment for nervous tissue function. BBB disruption is involved in many central nervous system diseases, including epilepsy. Evidence demonstrates that BBB breakdown may induce epileptic seizures, and conversely, seizure-induced BBB disruption may cause further epileptic episodes. This study was conducted based on the premise that the impairment of brain tissue during the triggering event may determine the organization and functioning of the brain during epileptogenesis, and that BBB may have a key role in this process. Our purpose was to investigate in rats the relationship between pilocarpine-induced status epilepticus (SE), and BBB integrity by determining the time course of the BBB opening and its subsequent recovery during the acute phase of the pilocarpine model. BBB integrity was assessed by quantitative and morphological methods, using sodium fluorescein and Evans blue (EB) dyes as markers of the increased permeability to micromolecules and macromolecules, respectively. Different time-points of the pilocarpine model were analyzed: 30 min after pilocarpine injection and then 1, 5, and 24 h after the SE onset. Our results show that BBB breakdown is a dynamic phenomenon and time-dependent, i.e., it happens at specific time-points of the acute phase of pilocarpine model of epilepsy, recovering in part its integrity afterwards. Pilocarpine-induced changes on brain tissue initially increases the BBB permeability to micromolecules, and subsequently, around 5 h after SE, the BBB breakdown to macromolecules occurs. After BBB breakdown, EB dye is captured by damaged cells, especially neurons, astrocytes, and oligodendrocytes. Although the BBB permeability to macromolecules is restored 24 h after the start of SE, the leakage of micromolecules persists and the consequences of BBB degradation are widely disseminated in the brain. Our findings reveal the existence of a temporal window of BBB dysfunction in the acute phase of the pilocarpine model that is important for the development of therapeutic strategies that could prevent the epileptogenesis.

Role of dorsal raphe nucleus GHS-R1a receptors in the regulation of inhibitory avoidance and escape behaviors in rats.

Ghrelin is a recently discovered peptide, mainly produced in the stomach and involved in body's energy-maintenance processes. Ghrelin exerts its actions by activating the growth hormone secretagogue receptor (GHS-R). Recent analyses indicate that ghrelin targets the brain to regulate a wealth of functions, including behavioral responses that have been associated with stress and anxiety mechanisms. In this context, evidence shows the presence of GHS-R receptors in the dorsal raphe nucleus (DRN), the main source of serotonergic neurons that innervate encephalic structures involved in emotional control. Our study aims to evaluate the effects of the pharmacological manipulation of ghrelin receptors located in the DRN on the expression of the behavioral responses of Wistar rats. Such responses were assessed in the elevated T maze (ETM), an experimental model that allows the measurement, in the same animal, of two defensive tasks, inhibitory avoidance and escape. Our results showed that the intra-DRN infusion of ghrelin impaired the acquisition of inhibitory avoidance, an anxiolytic-like effect, and facilitated the expression of escape response in the ETM, indicating a panicogenic-like effect. The intra-DRN administration of the ghrelin receptor (GHS-R1a) antagonist PF-04628935 did not alter the behavioral tasks assessed in the ETM. Finally, our results revealed that intra-DRN infusions of PF-04628935 prior to the administration of ghrelin into this area neutralized the behavioral effects obtained in the ETM. Taken together, our data reveal the involvement of DRN GHS-R1a receptors in the regulation of defensive tasks that have been associated with generalized anxiety and panic disorders.

Milk restriction or oligosaccharide supplementation in calves improves compensatory gain and digestive tract development without changing hormone levels.

We estimated the effect of oligosaccharide supplementation and feed restriction on calves. The study was divided into two experimental periods of 28 days each with 20 crossbred calves that had initial body weight of 37 Kg and housed in individual pens. The animals were split in four experimental groups: animals fed 6 L milk/day (CON) in the two periods, animals fed milk restricted (3 L milk/day) in the first period and followed by CON feeding in the second period (RES), animals receiving supplementation of 5 g/day of mannanoligosaccharide (MOS) and animals receiving supplementation of 5 g/day mannan and frutoligosaccharide (MFOS). At the end of the study, all the animals were slaughtered. The average weight gain was lower in the restricted group when compared with CON and MFOS groups in the first period (P < 0.05) and there were no difference among the groups in the second period. Animals supplemented with MOS showed a significant increases in jejunal villus height and rumen papillae, which were not observed for MFOS group (P < 0.05) compared with RES and CON groups. There were no difference in ghrelin and leptin levels among treatments during periods 1 and 2 (P > 0.05). Also, the expression of ghrelin receptors in the paraventricular region of the hypothalamus did not differ among groups. We conclude that milk restriction during the first weeks of life in calves resulted in compensatory gain and did not modify the hormonal profile and expression of the ghrelin receptor in the hypothalamus. Moreover, a prebiotic supplementation changed the development of intestinal and ruminal epithelium.

Long-term monotherapy treatment with vitamin E reduces oxidative stress, but not seizure frequency in rats submitted to the pilocarpine model of epilepsy.

The imbalance between antioxidant system and reactive oxygen species (ROS) generation is related to epileptogenesis, neuronal death, and seizure frequency. Treatment with vitamin E has been associated with neuroprotection and control of seizures. In most experimental studies, vitamin E treatment has short duration. Therefore, the aim of this study was to verify the role of long-term treatment with vitamin E in rats submitted to the pilocarpine model of epilepsy. Rats were divided into two main groups: control (Ctr) and pilocarpine (Pilo). Each one was subdivided according to treatment: vehicle (Ctr V and Pilo V) or vitamin E at dosages of 6 IU/kg/day (Ctr E6 and Pilo E6) or 60 IU/kg/day (Ctr E60 and Pilo E60). Treatment lasted 120 days from status epilepticus (SE). There were no statistical differences concerning treatment in the Ctr group for all variables, so the data were grouped. Carbonyl content in the hippocampus of Pilo V and Pilo E6 was higher compared with that of the Ctr group (8 ±â€¯1.5, 7.1 ±â€¯1, and 3.1 ±â€¯0.3 nmol carbonyl/mg protein, respectively for Pilo V, Pilo E6, and Ctr; p < 0.05). Carbonyl content was restored to control values in Pilo E60 rats (4.2 ±â€¯1.1 and 3.1 ±â€¯0.3 nmol carbonyl/mg protein, respectively for Pilo E60 and Ctr; p > 0.05). The volume of the hippocampal formation (6.5 ±â€¯0.3, 6.6 ±â€¯0.4, 6.3 ±â€¯0.3, and 7.4 ±â€¯0.2, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and subfields CA1 (1.6 ±â€¯0.1, 1.4 ±â€¯0.2, 1.5 ±â€¯0.1, and 2 ±â€¯0.05, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and CA3 (1.7 ±â€¯0.1, 1.5 ±â€¯0.2, 1.4 ±â€¯0.1, and 2 ±â€¯0.1, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) was reduced in the Pilo group regardless of treatment. Parvalbumin immunostaining was increased in the hilus of the Pilo E60 group compared with that in the Ctr group (26 ±â€¯2 and 39.6 ±â€¯8.3 neurons, respectively for Ctr and Pilo E60). No difference was found in seizure frequency and Neo-Timm staining. Therefore, long-term treatment with 60 IU/kg/day of vitamin E prevented oxidative damage in the hippocampus and increased hilar parvalbumin expression in rats with epilepsy without a reduction in seizure frequency.

Ghrelin potentiates cardiac reactivity to stress by modulating sympathetic control and beta-adrenergic response.

Prior evidence indicates that ghrelin is involved in the integration of cardiovascular functions and behavioral responses. Ghrelin actions are mediated by the growth hormone secretagogue receptor subtype 1a (GHS-R1a), which is expressed in peripheral tissues and central areas involved in the control of cardiovascular responses to stress. AIMS: In the present study, we assessed the role of ghrelin - GHS-R1a axis in the cardiovascular reactivity to acute emotional stress in rats. MAIN METHODS AND KEY FINDINGS: Ghrelin potentiated the tachycardia evoked by restraint and air jet stresses, which was reverted by GHS-R1a blockade. Evaluation of the autonomic balance revealed that the sympathetic branch modulates the ghrelin-evoked positive chronotropy. In isolated hearts, the perfusion with ghrelin potentiated the contractile responses caused by stimulation of the beta-adrenergic receptor, without altering the amplitude of the responses evoked by acetylcholine. Experiments in isolated cardiomyocytes revealed that ghrelin amplified the increases in calcium transient changes evoked by isoproterenol. SIGNIFICANCE: Taken together, our results indicate that the Ghrelin-GHS-R1a axis potentiates the magnitude of stress-evoked tachycardia by modulating the autonomic nervous system and peripheral mechanisms, strongly relying on the activation of cardiac calcium transient and beta-adrenergic receptors.

Hippocampal plasticity in rats submitted to a gastric restrictive procedure.

Bariatric surgery has been the most effective therapeutic intervention for morbidly obese patients. However, recent evidence has shown that this procedure may cause serious neurological complications such as Wernicke encephalopathy, depression, and memory impairment. With this in mind, we conducted an experimental study to investigate whether weight-reduction surgery would promote morphological changes in the hippocampal formation, a brain region linked to cognitive and emotional processes. To do so, the present study evaluated the hippocampal expression of parvalbumin interneurons in rats submitted to a gastric restrictive procedure (experimental phytobezoar). Our results demonstrated that rats with gastric-reduced capacity presented a significant increase in the expression of the parvalbumin interneurons in the hippocampal CA1 and CA3 subfields. These data are the first experimental evidence that restrictive bariatric surgery may alter hippocampal cytoarchitecture.

Expression of vitamin D receptor mRNA in the hippocampal formation of rats submitted to a model of temporal lobe epilepsy induced by pilocarpine.

Vitamin D (VD), is a steroid hormone with multiple functions in the central nervous system (CNS), producing numerous physiological effects mediated by its receptor (VDR). Clinical and experimental studies have shown a link between VD dysfunction and epilepsy. Along these lines, the purpose of our work was to analyze the relative expression of VDR mRNA in the hippocampal formation of rats during the three periods of pilocarpine-induced epilepsy. Male Wistar rats were divided into five groups: (1) control group; rats that received saline 0.9%, i.p. and were killed 7 days after its administration (CTRL, n=8), (2) SE group; rats that received pilocarpine and were killed 4h after SE (SE, n=8), (3) Silent group--7 days; rats that received pilocarpine and were killed 7 days after SE (SIL 7d, n=8), (4) Silent group--14 days; rats that received pilocarpine and were killed 14 days after SE (SIL 14d, n=8), (5) Chronic group; rats that received pilocarpine and were killed 60 days after the first spontaneous seizure, (chronic, n=8). The relative expression of VDR mRNA was determined by real-time PCR. Our results showed an increase of the relative expression of VDR mRNA in the SIL 7 days, SIL 14 days and Chronic groups, respectively (0.060+/-0.024; 0.052+/-0.035; 0.085+/-0.055) when compared with the CTRL and SE groups (0.019+/-0.017; 0.019+/-0.025). These data suggest the VDR as a possible candidate participating in the epileptogenesis process of the pilocarpine model of epilepsy.

Morte súbita nas epilepsias: resultados inspiradores revelados pela atividade física / Sudden unexpected death in epilepsy: sudden death risk decreased by physical activity

A epilepsia é a doença neurológica crônica grave mais comum e ofenômeno da morte súbita nas epilepsias (SUDEP) é uma das causasde morte mais comuns nesta síndrome neurológica. Várias propostastêm sido feitas com o intuito de esclarecer o fenômeno da SUDEP, e amaioria envolve o possível papel dos efeitos autonômicos, tais comodistúrbios cardiorrespiratórios, tendo recebido especial atenção. Nessesentido, estudos clínicos e experimentais têm demonstrado que aatividade física pode diminuir a freqüência de crises epilépticas, assimcomo melhorar a função cardiovascular e psicológica fisiológica dospacientes com epilepsia. Dessa forma, este artigo de revisão aborda o fenômeno da SUDEP e a possível contribuição da atividade física sobrepara a prevenção da SUDEP entre os pacientes com epilepsia.