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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.

Status epilepticus in immature rats leads to behavioural and cognitive impairment and epileptogenesis.

It remains under dispute whether status epilepticus (SE) in the perinatal period or early childhood or the underlying neuropathology is the cause of functional impairment later in life. The present study examined whether SE induced by LiCl–pilocarpine in normal immature brain (at the age of 12 or 25u2003days; P12 or P25) causes cognitive decline and epileptogenesis, and the data were compared to those of rats undergoing SE as adults. Rats in the P12 group had impaired memory (repeated exposure to open‐field paradigm) and emotional behaviour (lower proportion of open‐arm entries and higher incidence of risk assessment period in elevated plus‐maze) when assessed 3u2003months after SE, although not as severe as in the older age groups. Importantly, video‐electroencephalography monitoring 3u2003months after SE demonstrated that 25% of rats in the P12 and 50% in P25 group developed spontaneous seizures. Only nonconvulsive seizures (ictal activity in hippocampus accompanied by automatisms) were recorded in the P12 group whereas rats in the P25 group exhibited clonic convulsions. The present findings indicate that SE is harmful to the immature brain as early as P12, which might be compared with early infancy in humans.

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.

Long-term behavioral and morphological consequences of nonconvulsive status epilepticus in rats.

The aims of the present study were to ascertain whether nonconvulsive status epilepticus (NCSE) could give rise to long-term behavioral deficits and permanent brain damage. Two months after NCSE was elicited with pilocarpine (15 mg/kg i.p.) in LiCl-pretreated adult male rats, animals were assigned to either behavioral (spontaneous behavior, social interaction, elevated plus-maze, rotorod, and bar-holding tests) or EEG studies. Another group of animals was sacrificed and their brains were processed for Nissl and Timm staining as well as for parvalbumin and calbindin immunohistochemistry. Behavioral analysis revealed motor deficits (shorter latencies to fall from rotorod as well as from bar) and disturbances in the social behavior of experimental animals (decreased interest in juvenile conspecific). EEGs showed no apparent abnormalities. Quantification of immunohistochemically stained sections revealed decreased amounts of parvalbumin- and calbindin-immunoreactive neurons in the motor cortex and of parvalbumin-positive neurons in the dentate gyrus. Despite relatively inconspicuous manifestations, NCSE may represent a risk for long-term deficits.

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

Summary: u2002Purpose: 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.

Brain superoxide anion formation in immature rats during seizures: Protection by selected compounds

The widely-held assumption was that oxidative stress does not occur during seizures in the immature brain. The major finding of the present study concerns evidence of oxidative stress in the brain of immature rats during seizures induced by DL-homocysteic acid. Seizures were induced in 12-day-old rats by bilateral intracerebroventricular infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side) and oxidative stress was evaluated by in situ detection of superoxide anion (O(2)·(-)). Using hydroethidine (Het) method, the fluorescent signal of the oxidized products of Het (reflecting O(2)·(-) production) significantly increased (by 50%-60%) following 60 min lasting seizures in all the studied structures, namely CA1, CA3 and dentate gyrus of the hippocampus, cerebral cortex and thalamus. The enhanced O(2)·(-) production was substantially attenuated or completely prevented by substances providing an anticonvulsant effect, namely by a competitive NMDA receptor antagonist AP7, a highly selective and potent group II metabotropic glutamate receptor (mGluR) agonist 2R,4R-APDC and highly selective group III mGluR, subtype 8 agonist (S)-3,4-DCPG. Complete protection was achieved by two SOD mimetics Tempol and MnTMPYP which strongly suggest that the increased fluorescent signal reflects O(2)·(-) formation. In addition, both scavengers provided a partial protection against brain damage associated with the present model of seizures. Signs of neuronal degeneration, as evaluated by Fluoro-Jade B staining, were detected at 4h following the onset of seizures. The present findings thus suggest that the increased superoxide generation precedes neuronal degeneration and may thus play a causative role in neuronal injury. Occurrence of oxidative stress in brain of immature rats during seizures, as demonstrated in the present study, can have a clinical relevance for a novel approach to the treatment of epilepsy in children, suggesting that substances with antioxidant properties combined with the conventional therapies might provide a beneficial effect.

An Animal Model of Nonconvulsive Status Epilepticus: A Contribution to Clinical Controversies

Summary: u2002Purpose: To characterize electroencephalographic and behavioral effects as well as electrophysiologic and morphologic consequences of a subconvulsive dose of pilocarpine in lithium chloride–pretreated rats.

Changes in Cytochrome Oxidase in the Piriform Cortex after Status Epilepticus in Adult Rats

Summary:u2002 Purpose: The piriform cortex is involved in genesis and propagation of temporal lobe seizures. Degenerating neurons demonstrated by FluoroJade B staining are visible early after status epilepticus (SE) as well as after longer intervals. Furthermore, the piriform cortex is activated during an early phase of experimental temporal seizures, as described by magnetic resonance imaging (MRI) studies. It indicates that the early activity of the piriform cortex should be accompanied by increased adenosine triphosphate (ATP) production. Cytochrome oxidase activity in the brain may be used as an endogenous metabolic marker for neurons. The present research studied activity of the cytochrome oxidase separately in the rostral and caudal parts of the piriform cortex after lithium chloride–pilocarpine–induced SE in adult rats.

Effect of free radical spin trap N-tert-butyl-α-phenylnitrone (PBN) on seizures induced in immature rats by homocysteic acid

The present study has examined the effect of free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) 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). PBN was given i.p. in two doses (100 mg/kg each), 30 min prior and 30 min after dl-HCA infusion. PBN did not significantly influence the severity of seizures, evident both from the behavioral symptoms and EEG recordings. PBN normalized decreased ATP levels in the hippocampus, occurring during the acute phase of seizures ( approximately 45-50 min after infusion) and persisting until the end of the 24-h recovery period. PBN also led to normalization of decreased glucose levels and to a significant reduction of lactate accumulation in the cerebral cortex and hippocampus. The neuroprotective effect of PBN was evaluated after 24 h and 6 days of survival following dl-HCA-induced seizures (Nissl and Fluoro-Jade B staining). The administration of PBN resulted in a partial amelioration of severe damage observed in many brain regions following infusion of dl-HCA alone. The data suggest that increased free radical production is apparently occurring during seizures induced in immature rats by homocysteic acid. Free radical scavenger PBN had a clear-cut protective effect, evident as the improved recovery of brain energy status and as a partial, but significant, attenuation of neuronal degeneration associated with this model of seizures.