Maria Carolina Doretto

An effective anticonvulsant prepared following a host–guest strategy that uses hydroxypropyl-β-cyclodextrin and benzaldehyde semicarbazone☆

The convulsions of approximately 25% of epileptics are inadequately controlled by currently available medication; therefore the preparation of new antiepileptic drugs is of great interest. Aryl semicarbazones can be considered a new class of compounds presenting anticonvulsant activity. In addition, they can be orally administered and are more active as anticonvulsants than mephenytoin or phenobarbital. However, one disadvantage of these compounds is their low water solubility. As a strategy to circumvent this problem, a 1:1 inclusion compound of benzaldehyde semicarbazone (BS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was prepared and characterized. The anticonvulsant activities of the free semicarbazone and of the inclusion compound were evaluated in rats using the maximum electroshock and audiogenic seizures screenings. In both tests the minimum dose of compound necessary to produce activity decreases from 100mg/kg for the free semicarbazone to 35 mg/kg for the inclusion compound, indicating a significant increase in the bio-availability of the drug.

Distinct patterns of electrical stimulation of the basolateral amygdala influence pentylenetetrazole seizure outcome.

Our working hypothesis is that constant interpulse interval (IPI) electrical stimulation would resonate with endogenous epileptogenic reverberating circuits, inducing seizures, whereas a random interinterval electrical stimulation protocol would promote desynchronization of such neural networks, producing an anticonvulsant effect. Male Wistar rats were stereotaxically implanted with a bipolar electrical stimulation electrode in the amygdala. Pentylenetetrazole (10mg/ml/min) was continuously infused through an intravenous catheter to induce seizures while four different patterns of temporally coded electrical stimulation were applied: periodic stimulation (PS), pseudo-randomized IPI stimulation (LH), restrictively randomized IPI stimulation (IH), and bursts of 20-ms IPIs (burst). PS decreased the pentylenetetrazole threshold to forelimb clonus, whereas IH increased the threshold to forelimb clonus and to generalized tonic-clonic seizures. We hypothesize that PS facilitates forelimb clonus by reverberating with epileptogenic circuits in the limbic system, whereas IH delays forelimb clonus and generalized tonic-clonic seizures by desynchronizing the epileptic neural networks in the forebrain-midbrain-hindbrain circuits.

Assessment of the seizure susceptibility of Wistar Audiogenic rat to electroshock, pentyleneterazole and pilocarpine.

This work evaluates the seizure susceptibility of nai;ve female Wistar Audiogenic rats (WARs), a genetic model of reflex epilepsy in which seizures are induced by high-intensity sound stimulation (120 dB SPL), to other pro-convulsive stimuli: transauricular electroshock (ES), pentylenetetrazole (PTZ) and pilocarpine (PILO). Normal Wistar rats from the main breeding stock of the Institute of Biological Sciences, UFMG were taken as controls. Electroshock seizures were induced through a pair of ear-clip electrodes (10 mA, at a frequency of 60 Hz, applied for 1 s). In order to test WAR susceptibility to chemically induced seizures, animals were treated either with PTZ (37.5 mg/kg i.p.) or PILO (200, 270 and 300 mg/kg i.p.). Seizure severity was evaluated by appropriate behavioral severity index scales (SI) specific to each epilepsy model and tested for statistical significance using the non-parametric Mann-Whitney Rank Sum test. Results show a significantly greater susceptibility of WARs for ES (SI(WAR)=3+/-3/3, SI(Wistar)=1+/-1/1; median+/-interquartile range 25%/75%, P<0.01) and PTZ (SI(WAR)=4+/-4/4, SI(Wistar)=1+/-1/4; median+/-interquartile range 25%/75%, P<0.02), as indicated by significantly higher SI scores and shorter latencies for seizure onset (T(WAR)=71+/-7 s, T(Wistar)=94+/-8 s; P<0.05 Student t-test, mean+/-S.E.M.). Although PILO also caused higher SI scores in WARs (WAR(200 mg)=1+/-1/1, Wistar(200 mg)=1+/-1/1; WAR(270 mg)=1.5+/-1/2, Wistar(270 mg)=1+/-1/1.25; WAR(300 mg)=9+/-1/9, Wistar(300 mg)=4+/-1.5/7.5; median+/-interquartile range 25%/75%), statistically significant differences were not observed. In conclusion, our results show that WARs have an inherited broader predisposition for seizures.

Involvement of prolactin, vasopressin and opioids in post-ictal antinociception induced by electroshock in rats.

The neurochemical mechanisms involved in post-ictal antinociception remain to be elucidated. Application of electroconvulsive shock (ECS) to rats results in post-ictal antinociception. The objective of this study was to identify endogenous substances that could participate in antinociception during post-ictal depression induced by ECS (70 mA, 60 Hz, 1 s). Antinociception was measured by the rat paw-pressure test, in which increased sensitivity is induced by intraplantar injection of carrageenan. This test proved to be efficient in detecting the electroshock-induced antinociception. Intense post-ictal antinociception was observed over a period of 30 min after the end of the seizure. It was used nonspecific opioid and specific vasopressin antagonists and the prolactin (PRL) release inhibitor to test the reversal of antinociception. Administration of naloxone (5, 7.5 and 10 mg/kg) blocked the post-ictal antinociception. The V(1) (125 microg/kg) and V(2) (250 microg/kg) vasopressin receptor antagonists ([beta-mercapto-beta,beta-cyclopentamethylenepropionyl(1),O-Et-Tyr(2),Val(4),Arg(8)]-vasopressin and [adamantaneacetyl(1),O-Et-d-Tyr(2),Val(4),Abu(6),Arg(8,9)]-vasopressin) also inhibited the nociceptive response. The antinociception blockade was more intense after administration of the V(1) receptor antagonist. Bromocriptine (4, 8 and 12 mg/kg) was able to reverse antinociception behavior during the post-ictal period. Morphine (1, 2 and 4 mg/kg), vasopressin (12.5, 100 and 400 microg/kg) and prolactin (100, 200 and 400 microg/kg) administration promoted a higher nociceptive threshold. It was administered the three substances with their respective antagonists to verify the opioidergic pathway and vasopressin and prolactin release interactions, and as a positive control. We observed that the tested mediators were released in an independent manner, indicating no interference in which other.

Inclusion of Benzaldehyde Semicarbazone into β-Cyclodextrin Produces a Very Effective Anticonvulsant Formulation

Aryl semicarbazones can be considered a novel class of compounds presenting anticonvulsant activity. In order to improve the efficiency and bioavailability of benzaldehyde semicarbazone (BS) we used the host–guest strategy and β-cyclodextrin (β-CD) to prepare a BS/β-CD 1:1 inclusion compound, which was characterized by thermal analyses (TG, DTG, DSC), XRD powder pattern diffraction analyses, infrared data and 1H, 13C, 2D-ROESY NMR and 1H relaxation times. The BS/β-CD inclusion compound was tested in rats using the maximum electroshock (MES) screen. The minimum dose necessary to produce anticonvulsant activity decreased from 100 mg/Kg (ip or vo) for the free semicarbazone to 25 mg/Kg/vo (75%) and 15 mg/Kg/ip (85%), indicating that the host–guest strategy that uses β-CD and BS is very effective and could be successfully employed in the preparation of pharmaceutical formulationof anticonvulsants.

Cardiac dysfunction in rats prone to audiogenic epileptic seizures.

PURPOSE Cardiac dysfunction is one of the possible causes of sudden unexpected death in epilepsy (SUDEP). Therefore, the objective of this study was to evaluate cardiac and electrocardiographic parameters in rats with audiogenic epileptic seizures (WAR--Wistar audiogenic rats). METHODS In vivo arterial pressure, heart rate (HR), autonomic tone and electrocardiography (ECG) were measured in awake animals in order to examine cardiac function and rhythm. Ex vivo, the Langendorff technique was used to analyze the cardiac function and the severity of reperfusion arrhythmias. In vitro, confocal microscopy was used to evaluate calcium transient parameters of isolated ventricular cardiomyocytes. RESULTS In vivo autonomic tone evaluation revealed enhanced sympathetic activity, changes in cardiac function with increased systolic arterial pressure and higher basal HR in WAR. In addition, ECG analysis demonstrated electrical alterations with prolongation of the QT interval and QRS complex in these animals. Ex vivo, we observed a decrease in systolic tone and HR and an increase in the duration of ischemia/reperfusion arrhythmias in WAR. Moreover, intracellular Ca2+ handling analysis revealed an increase in the peak of calcium and calcium transient decay in audiogenic rats. Treatment with atenolol (β1-adrenergic antagonist) normalized the systolic tone, reduced cardiac hypertrophy and the associated increase in the susceptibility to reperfusion arrhythmias observed in WAR. CONCLUSION We present evidence that chronic disturbances in sympathetic tone in WAR cause increases the risk to life-threatening arrhythmias. Our results support a relationship between seizures, cardiac dysfunction and cardiac arrhythmias, which may contribute to the occurrence of SUDEP.

Evidence for augmented brainstem activated forebrain seizures in Wistar Audiogenic Rats subjected to transauricular electroshock

Previous work from our laboratory has shown that naïve Wistar Audiogenic Rats (WARs), a genetic model of reflex epilepsy in which seizures are induced by high-intensity sound stimulation (120 dB SPL), are seizure-prone to a variety of pro-convulsive stimuli (e.g., transauricular electroshock, pentylenetetrazole and pilocarpine). On the other hand, repetitive acoustic stimulation of WARs causes a slow recruitment of limbic structures, known as audiogenic kindling, changing seizure expression to include behavior characteristic of temporal-lobe epilepsy. Thus, our hypothesis is that WARs have facilitated acoustic-limbic projections when compared to Wistar controls. Wistar controls (n = 9) and WARs (n = 9) underwent EEG electrode implants in the cortex-Cx, amygdaloid complex-AMY and inferior colliculus-IC and received one low current transauricular electrical stimulus (ES) daily, for three consecutive days, with intensities of 10, 20 and 30 mA, respectively. The video-electroencephalographic activity was recorded 1 min before and 4 min after ES. Our results confirm previously described data indicating a greater susceptibility of WARs to seizure. However, low current ES (e.g., 20 mA) triggered epileptiform activity in the AMY only after epileptiform EEG was visible in the Cx and IC electrode leads. The AMY after-discharge continued even though no evident epileptiform activity was present in the Cx. In conclusion, our results add electrophysiological data to previously published behavioral evidence of WAR enhanced susceptibility to ES seizures and, also, support the hypothesis that the acoustic-limbic circuitry is facilitated even in unkindled WARs.

Do endogenous opioids and nitric oxide participate in the anticonvulsant action of dipyrone

It was previously reported that systemic administration of dipyrone inhibited the tonic component of generalized tonic-clonic seizures in both the electroshock and the audiogenic seizure models. The aim of the present study was to investigate the mechanisms involved in the anticonvulsant action of dipyrone by assessing the role of nitric oxide and opioids in the electroshock (female 60- to 90-day-old Wistar rats, N = 5-11) and audiogenic seizure (female 60- to 90-day-old Wistar audiogenic rats, N = 5-11) models of epilepsy. Naloxone (5 mg/kg, sc) significantly reversed the anticonvulsant effect of dipyrone in rats submitted to the induction of audiogenic seizures (ANOVA/Bonferronis test), suggesting the involvement of opioid peptides in this action. In the electroshock model no reversal of the anticonvulsant effect of dipyrone by naloxone (5 mg/kg, sc) was demonstrable. The acute (120 mg/kg, ip) and chronic (25 mg/kg, ip, twice a day/4 days) administration of L-NOARG did not reverse the anticonvulsant action of dipyrone in the audiogenic seizure model, suggesting that the nitric oxide pathway does not participate in such effect. Indomethacin (10, 20 and 30 mg/kg, ip) used for comparison had no anticonvulsant effect in the audiogenic seizure model. In conclusion, opioid peptides but not nitric oxide seem to be involved in the anticonvulsant action of dipyrone in audiogenic seizures.

Wistar Audiogenic Rats (WAR) exhibit altered levels of cytokines and brain-derived neurotrophic factor following audiogenic seizures

Increasing body of evidence suggests that inflammatory and neurotrophic factors might be important for epileptogenesis. Most animal studies demonstrated altered levels of these mediators in drug-induced models of seizures and epilepsy. In the present study, we investigated the production of cytokines and a neurotrophin in the brain of Wistar Audiogenic Rats (WAR), a genetic model of epilepsy, stimulated with high-intensity sound. Four hours after stimulation, animals were decapitated and the hippocampus, inferior colliculus, striatum and cortex were removed for evaluation of the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and brain derived neurotrophic factor (BDNF). All the cytokines and BDNF levels were increased in the cortex. Increased levels of TNF-α and IL-6 were also observed in the striatum. Finally, TNF-α also increased in the inferior colliculus after the seizures induced by high-intensity sound. Although different studies have demonstrated that the levels of cytokines and BDNF increase in animal models of epilepsy induced by chemical stimuli, we provided here evidence that these mediators are also increased in WAR, a genetic model of epilepsy. Thus, the observed increase in these mediators might be involved in the pathophysiology of epilepsy.

Cardiovascular dysautonomia after seizures induced by maximal electroshock in Wistar rats

BACKGROUND Cardiac dysfunction is one of the possible causes of sudden unexpected death in epilepsy (SUDEP). Therefore, it was the objective of this study to evaluate the cardiac and electrocardiographic parameters after seizures induced by maximal electroshock (MES) in Wistar rats. METHODS Electroshock seizures were induced in Wistar rats through a pair of ear-clip electrodes (10 mA at a frequency of 60 Hz applied for one second). In vivo electrocardiography (ECG) was performed in awake animals for analysis of heart rate variability (HRV) and cardiac rhythm. Ex vivo the Langendorff technique was used to analyze cardiac function and observe the incidence and severity of reperfusion arrhythmias. RESULTS Convulsive seizures triggered by MES induced profound abnormalities in cardiac rhythm with serious electrocardiographic changes including ST-elevation, bundle branch block, atrioventricular nodal escape rhythm and premature ventricular contractions. ECG analysis demonstrated a consistent period of postictal bradyarrhythmia resulting in a transiently irregular cardiac rhythm with highly variable and prolonged QRS complexes and RR, PR, QT and QTc intervals. HRV evaluation revealed an increase in the high-frequency range of the power, suggesting an imbalance in the autonomic control of the heart with a postictal enhancement of parasympathetic tone. In addition, we observed in isolated heart a decrease in systolic tone and an increase in the coronary flow, heart rate and incidence/duration of ischemia-reperfusion arrhythmias. CONCLUSION The present study supports a relationship betweem seizures, cardiac dysfunction and cardiac arrhythmias. This relationship may partially account for the occurrence of SUDEP.