Renata Haugvicová

Status Epilepticus Causes Necrotic Damage in the Mediodorsal Nucleus of the Thalamus in Immature Rats

Status epilepticus (StE) in immature rats causes long-term functional impairment. Whether this is associated with structural alterations remains controversial. The present study was designed to test the hypothesis that StE at an early age results in neuronal loss. StE was induced with lithium–pilocarpine in 12-d-old rats, and the presence of neuronal damage was investigated in the brain from 12 hr up to 1 week later using silver and Fluoro-Jade B staining techniques. Analysis of the sections indicated consistent neuronal damage in the central and lateral segments of the mediodorsal nucleus of the thalamus, which was confirmed using adjacent cresyl violet-stained preparations. The mechanism of thalamic damage (necrosis vs apoptosis) was investigated further using TUNEL, immunohistochemistry for caspase-3 and cytochrome c, and electron microscopy. Activated microglia were detected using OX-42 immunohistochemistry. The presence of silver and Fluoro-Jade B-positive degenerating neurons in the mediodorsal thalamic nucleus was associated with the appearance of OX-42-immunopositive activated microglia but not with the expression of markers of programmed cell death, caspase-3, or cytochrome c. Electron microscopy revealed necrosis of the ultrastructure of damaged neurons, providing further evidence that the mechanism of StE-induced damage in the mediodorsal thalamic nucleus at postnatal day 12 is necrosis rather than apoptosis. Finally, these data together with previously described functions of the medial and lateral segments of the mediodorsal thalamic nucleus suggest that some functions, such as adaptation to novelty, might become compromised after StE early in development.

Behavioral and metabolic changes in immature rats during seizures induced by homocysteic acid : the protective effect of NMDA and non-NMDA receptor antagonists

Bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA) (600 nmol on each side) to immature 12-day-old rats induced generalized clonic-tonic seizures, recurring frequently for at least 90 min, with a high rate of survival. Electrographic recordings from sensorimotor cortex, hippocampus, and striatum demonstrated isolated spikes in the hippocampus and/or striatum as the first sign of dl-HCA action. Generalization of epileptic activity occurred during generalized clonic-tonic seizures, but electroclinical correlation was very low; dissociation between EEG pattern and motor phenomena was common. Seizures were accompanied by large decreases of cortical glucose and glycogen and by approximately 7- to 10-fold accumulation of lactate. ATP and phosphocreatine (PCr) levels remained unchanged even during longlasting (3 h) convulsions. Metabolite levels became normalized during the recovery period (24 h). The examination of the effect of selected antagonists of NMDA [AP7 (18.5 and 37 mg/kg, respectively), MK-801 (0.5 mg/kg)] and non-NMDA [NBQX (10, 15 and 30 mg/kg, respectively)] receptors revealed that seizures could be attenuated or prevented (depending on the dose employed) by antagonists of both NMDA and non-NMDA receptors, as evaluated not only according to the suppression of behavioral manifestations of seizures, but also in terms of the protection of metabolite changes accompanying seizures. All antagonists employed, when given alone in the same doses as those used for seizure protection, did not influence metabolite levels, with the exception of increased glucose concentrations. Furthermore, the pronounced anticonvulsant effect could be achieved by the combined treatment with low subthreshold doses of NMDA (AP7) and non-NMDA (NBQX) receptor antagonists, which may be of potential significance for a new approach to the treatment of epilepsy.

Neuronal Cell Death in Hippocampus Induced by Homocysteic Acid in Immature Rats

Summary:  Purpose: To examine the morphologic alterations in the cerebral cortex and hippocampus of immature rats 6 days after the generalized clonic–tonic seizures induced by homocysteic acid (HCA).

Convulsant Action of D,L-Homocysteic Acid and Its Stereoisomers in Immature Rats

Summary: Purpose: We wished to characterize the convulsant effect of homocysteic acid (HCA) in developing rats.

Seizures induced in immature rats by homocysteic acid and the associated brain damage are prevented by group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate.

The present study has examined the anticonvulsant and neuroprotective effect of group II metabotropic glutamate receptor (mGluR) agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) in the model of seizures induced in immature 12-day-old rats by bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the pups which had received 2R,4R-APDC. Low doses of 2R,4R-APDC (0.05 nmol/side) provided a pronounced anticonvulsant effect which was abolished by pretreatment with a selective group II mGluR antagonist LY341495. Generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). EEG recordings support the marked anticonvulsant effect of 2R,4R-APDC, nevertheless, this was only partial. In spite of the absence of obvious motor phenomena, isolated spikes or even short periods of partial ictal activity could be observed. Isolated spikes could also be seen in some animals after application of 2R,4R-APDC alone, reflecting most likely subclinical proconvulsant activity of this agonist. The neuroprotective effect of 2R,4R-APDC was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration, as revealed by Fluoro-Jade B staining, was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas 2R,4R-APDC pretreatment provided substantial neuroprotection. The present findings support the possibility that group II mGluRs are a promising target for a novel approach to treating epilepsy.

Sustained deficiency of mitochondrial complex I activity during long periods of survival after seizures induced in immature rats by homocysteic acid

Our previous work demonstrated the marked decrease of mitochondrial complex I activity in the cerebral cortex of immature rats during the acute phase of seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side) and at short time following these seizures. The present study demonstrates that the marked decrease ( approximately 60%) of mitochondrial complex I activity persists during the long periods of survival, up to 5 weeks, following these seizures, i.e. periods corresponding to the development of spontaneous seizures (epileptogenesis) in this model of seizures. The decrease was selective for complex I and it was not associated with changes in the size of the assembled complex I or with changes in mitochondrial content of complex I. Inhibition of complex I was accompanied by a parallel, up to 5 weeks lasting significant increase (15-30%) of three independent mitochondrial markers of oxidative damage, 3-nitrotyrosine, 4-hydroxynonenal and protein carbonyls. This suggests that oxidative modification may be most likely responsible for the sustained deficiency of complex I activity although potential role of other factors cannot be excluded. Pronounced inhibition of complex I was not accompanied by impaired ATP production, apparently due to excess capacity of complex I documented by energy thresholds. The decrease of complex I activity was substantially reduced by treatment with selected free radical scavengers. It could also be attenuated by pretreatment with (S)-3,4-DCPG (an agonist for subtype 8 of group III metabotropic glutamate receptors) which had also a partial antiepileptogenic effect. It can be assumed that the persisting inhibition of complex I may lead to the enhanced production of reactive oxygen and/or nitrogen species, contributing not only to neuronal injury demonstrated in this model of seizures but also to epileptogenesis.

Mitochondrial complex I inhibition in cerebral cortex of immature rats following homocysteic acid-induced seizures

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60-90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

Seizures induced by homocysteic acid in immature rats are prevented by group III metabotropic glutamate receptoragonist (R,S)-4-phosphonophenylglycine

The potential anticonvulsant effect of group III metabotropic glutamate receptor (mGluR) agonist (R,S)-4-phosphonophenylglycine ((R,S)-PPG) against seizures induced in immature 12-day-old rats by bilateral intracerebroventricular (icv) infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side) was examined in the present study. Rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45 to 50 min after infusion. Comparable time intervals were used for sacrificing the pups which had received (R,S)-PPG. Low doses of (R,S)-PPG (10 nmol, icv) provided a pronounced anticonvulsant effect which was abolished by pretreatment with a selective group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate. Generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (glucose and glycogen decreases) or markedly ameliorated (an accumulation of lactate). Despite the absence of obvious motor phenomena, EEG recordings revealed sporadic ictal activity, mostly in the dorsal hippocampus. Spreading of this activity into the frontal cortex was rather exceptional. The latency of ictal EEG in pretreated rats was significantly prolonged. Our data suggest that the predominant effect of (R,S)-PPG might concern seizure spread. The administration of (R,S)-PPG alone did not cause any overt behavioral side effects; it did not change the EEG pattern and did not influence cortical metabolite levels, with the exception of increased concentrations of glucose. The present findings suggest that group III mGlu receptor agonists may be of therapeutic significance for treating childhood epilepsies.

Dynamic Changes of Status Epilepticus-Induced Neuronal Degeneration in the Mediodorsal Nucleus of the Thalamus During Postnatal Development of the Rat

Summary:  Purpose: Status epilepticus (SE) was previously found to induce damage in the mediodorsal nucleus of the thalamus (MD) in both adult and immature rats. This study was designed to describe age‐related changes of SE‐induced neuronal degeneration in this part of the brain.

Anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine against seizures induced in immature rats by homocysteic acid

The present study has examined the anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG), a highly selective agonist for subtype 8 of group III metabotropic glutamate receptors (mGluRs), against seizures induced in immature 12-day-old rats by bilateral icv infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the animals which had received (S)-3,4-DCPG (0.25 nmol/each side, 15-20 min prior to infusion of DL-HCA or saline). This agonist provided a pronounced anticonvulsant effect, generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). Anticonvulsant effect of (S)-3,4-DCPG was also evident from the EEG recordings, nevertheless, it was not complete. In spite of the absence of obvious motor phenomena, sporadic ictal activity could be seen in some animals. Isolated spikes could also be observed in some animals after administration of (S)-3,4-DCPG alone. The neuroprotective effect of (S)-3,4-DCPG was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas pretreatment with (S)-3,4-DCPG provided substantial neuroprotection. The present findings suggest that receptor subtype 8 of group III mGluRs may be considered a promising target for drug therapy in childhood epilepsies in the future.