Claude G. Wasterlain

Pathophysiological mechanisms of brain damage from status epilepticus

Summary: Human status epilepticus (SE) is consistently associated with cognitive problems, and with widespread neuronal necrosis in hippocampus and other brain regions. In animal models, convulsive SE causes extensive neuronal necrosis. Nonconvulsive SE in adult animals also leads to widespread neuronal necrosis in vulnerable regions, although lesions develop more slowly than they would in the presence of convulsions or anoxia. In very young rats, nonconvulsive normoxic SE spares hippocampal pyramidal cells, but other types of neurons may not show the same resistance, and inhibition of brain growth, DNA and protein synthesis, and of myelin formation and of synaptogenesis may lead to altered brain development. Lesions induced by SE may be epileptogenic by leading to misdirected regeneration. In SE, glutamate, aspartate, and acetylcholine play major roles as excitatory neurotransmitters, and GABA is the dominant inhibitory neurotransmitter. GABA metabolism in substantia nigra (SN) plays a key role in seizure arrest. When seizures stop, a major increase in GABA synthesis is seen in SN postictally. GABA synthesis in SN may fail in SE. Extrasynaptic factors may also play an important role in seizure spread and in maintaining SE. Glial immaturity, increased electrotonic coupling, and SN immaturity facilitate SE development in the immature brain. Major increases in cerebral blood flow (CBF) protect the brain in early SE, but CBF falls in late SE as blood pressure falters. At the same time, large increases in cerebral metabolic rate for glucose and oxygen continue throughout SE. Adenosine triphosphate (ATP) depletion and lactate accumulation are associated with hypermetabolic neuronal necrosis. Excitotoxic mechanisms mediated by both N‐methyl‐d‐aspartate (NMDA) and non‐NMDA glutamate receptors open ionic channels permeable to calcium and play a major role in neuronal injury from SE. Hypoxia, systemic lactic acidosis, CO2 narcosis, hyperkalemia, hypoglycemia, shock, cardiac arrhythmias, pulmonary edema, acute renal tubular necrosis, high output failure, aspiration pneumonia, hyperpyrexia, blood leukocytosis and CSF pleocytosis are common and potentially serious complications of SE. Our improved understanding of the pathophysiology of brain damage in SE should lead to further improvement in treatment and outcome.

Trafficking of GABA(A) receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus.

During status epilepticus (SE), GABAergic mechanisms fail and seizures become self-sustaining and pharmacoresistant. During lithiumpilocarpine-induced SE, our studies of postsynaptic GABAA receptors in dentate gyrus granule cells show a reduction in the amplitude of miniature IPSCs (mIPSCs). Anatomical studies show a reduction in the colocalization of the β2/β3 and γ2 subunits of GABAA receptors with the presynaptic marker synaptophysin and an increase in the proportion of those subunits in the interior of dentate granule cells and other hippocampal neurons with SE. Unlike synaptic mIPSCs, the amplitude of extrasynaptic GABAA tonic currents is augmented during SE. Mathematical modeling suggests that the change of mIPSCs with SE reflects a decrease in the number of functional postsynaptic GABAA receptors. It also suggests that increases in extracellular [GABA] during SE can account for the tonic current changes and can affect postsynaptic receptor kinetics with a loss of paired-pulse inhibition. GABA exposure mimics the effects of SE on mIPSC and tonic GABAA current amplitudes in granule cells, consistent with the model predictions. These results provide a potential mechanism for the inhibitory loss that characterizes initiation of SE and for the pharmacoresistance to benzodiazepines, as a reduction of available functional GABAA postsynaptic receptors. Novel therapies for SE might be directed toward prevention or reversal of these losses.

Apoptosis in a Neonatal Rat Model of Cerebral Hypoxia-Ischemia

BACKGROUND AND PURPOSE The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. METHODS Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. RESULTS The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. CONCLUSIONS Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.

Time dependent decrease in the effectiveness of antiepileptic drugs during the course of self-sustaining status epilepticus

An animal model of self-sustaining status epilepticus (SSSE) induced in rats by brief intermittent perforant path stimulation (PPS) was examined with regard to the effects of two conventional antiepileptic drugs, diazepam and phenytoin. Thirty or sixty minutes PPS induced SSSE characterized by continuous behavioral and electrographic seizures lasting for hours. Both diazepam (10 mg/kg i. v.) and phenytoin (50 mg/kg i.v.) prevented the establishment of SSSE when administered 10 min prior to PPS. The injection of diazepam to seizing animals, 10 min after the end of 30 min PPS, was significantly less effective than pretreatment in attenuating SSSE. Administration of diazepam after 60 min PPS was characterized by a further decrease of its efficacy. Phenytoin was effective in aborting SSSE when injected 10 min after 30 min PPS. However, its efficacy was vastly decreased if injected 40 min after 30 min PPS, or 10 min after 60 min PPS. It is concluded that antiepileptic drugs, while highly effective in blocking the induction of SSSE, failed to affect its maintenance. SSSE induced by PPS is an advantageous animal model of refractory status epilepticus, which may be used in preclinical studies of novel antiepileptic drugs.

Dentate granule cells form novel basal dendrites in a rat model of temporal lobe epilepsy

Mossy fibre sprouting and re-organization in the inner molecular layer of the dentate gyrus is a characteristic of many models of temporal lobe epilepsy including that induced by perforant-path stimulation. However, neuroplastic changes on the dendrites of granule cells have been less-well studied. Basal dendrites are a transient morphological feature of rodent granule cells during development. The goal of the present study was to examine whether granule cell basal dendrites are generated in rats with epilepsy induced by perforant-path stimulation. Adult Wistar rats were stimulated for 24 h at 2 Hz and with intermittent (1/min) trains (10 s duration) of single stimuli at 20 Hz (20 V, 0.1 ms) delivered 1/min via an electrode placed in the angular bundle. The brains of these experimental rats and age- and litter-matched control animals were processed for the rapid Golgi method. All rats with perforant-path stimulation displayed basal dendrites on many Golgi-impregnated granule cells. These basal dendrites mainly originated from their somata at the hilar side and then extended into the hilus. Quantitative analysis of more than 800 granule cells in the experimental and matched control brains showed that 6-15% (mean=8.7%) of the impregnated granule cells have spiny basal dendrites on the stimulated side, as well as the contralateral side (mean=3.1%, range=2.9-3.9%) of experimental rats, whereas no basal dendrites were observed in the dentate gyrus from control animals. The formation of basal dendrites appears to be an adaptive morphological change for granule cells in addition to the previously described mossy fibre sprouting, as well as dendritic and somatic spine formation observed in the dentate gyrus of animal and human epileptic brains. The presence of these dendrites in the subgranular region of the hilus suggests that they may be postsynaptic targets of the mossy fibre collaterals.

Posthypoxic treatment with MK‐801 reduces hypoxic‐ischemic damage in the neonatal rat

We evaluated the neuroprotective effect of MK-801, a noncompetitive, selective N-methyl-D-aspartate receptor antagonist, in a neonatal hypoxic-ischemic animal model. Seven-day-old rats underwent bilateral ligation of the carotid arteries followed by exposure to an 8% oxygen atmosphere for 1 hr. We sacrificed the animals 72 hrs later and assessed the hypoxic-ischemic brain damage histologically. MK-801 (10 mg/kg), administered IP 0.5 hr before the hypoxia, completely prevented hypoxic-ischemic infarction in cerebral cortex, while treatment immediately and 1 hr after the end of the hypoxia resulted in 76% and 52% reduction in the infarcted area, respectively. MK-801, given 0.5 hr before and immediately after the insult, reduced striatal damage and, given 0.5 hr before, attenuated neuronal necrosis in hippocampal regions. These results show that in neonates MK-801 is neuroprotective even when administered up to 1 hr after the end of a hypoxic-ischemic insult.

N-methyl-d-asparate receptor antagonists abolish the maintenance phase of self-sustaining status epilepticus in rat

We examined the effects of blockers of N-methyl-D-asparate (NMDA) and +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors on the maintenance of self-sustaining status epilepticus (SSSE) induced in rats by brief intermittent electrical stimulation of the perforant path (PPS). Blocking of NMDA receptor at the PCP site by MK-801 (0.5 mg/kg, i.p.) or ketamine (10 mg/kg, i.p.) as well as at the glycine allosteric site by intrahippocampal 5,7-dichlorokynurenic acid (5,7-DCK, 10 nmol), rapidly and irreversibly aborted both behavioral and electrographic manifestation of SSS. Intrahippocampal injection of the AMPA/kainate receptor blocker 6-cyano7-nitroquinixaline-3-dione (CNQX, 10 nmol) transiently suppressed seizures, which reappeared 4-5 h later. We suggest that the maintenance phase of SSSE depends on activation of NMDA receptors and that NMDA receptor blockers may be a promising class of compounds for the treatment of status epilepticus.

Effects of neonatal status epilepticus on rat brain development

A single, 2-hour episode of status epilepticus induced by flurothyl (1,500 μl) in 4-day-old rats irreversibly curtailed brain weight and brain DNA. Status epilepticus inhibited DNA synthesis but did not increase DNA breakdown and produced no histologic lesions. Rats with status epilepticus showed delayed behavioral milestones and reduced seizure thresholds several weeks after status. After milder convulsions (flurothyl 750

Epileptogenesis after status epilepticus reflects age- and model-dependent plasticity

mUl, bicuculline), brain DNA was curtailed at 7 days but returned to normal at 30 days. These results suggest that, in the immature brain, epileptic seizures too mild to cause cell necrosis can inhibit DNA synthesis and permanently curtail brain DNA content. This may account for the great vulnerability of the immature brain to epileptic seizures.

Anticonvulsant activity of a nonpeptide galanin receptor agonist

Although epilepsy often begins in childhood, factors that contribute to the development of epilepsy as a consequence of status epilepticus (SE) during early development are poorly understood. We investigated animal models in which seizure‐induced epileptogenicity could be studied. Rats undergoing self‐sustaining SE induced by perforant path stimulation (PPS) at the ages of postnatal day 21 (P21) and P35 were compared with those subjected to SE by lithium and pilocarpine (LiPC). Although only one animal subjected to PPS at P21 developed chronic spontaneous seizures by several months of observation, all the animals subjected to PPS at P35 became epileptic. In the LiPC model, however, most of the rat pups subjected to SE at P21 became epileptic. Animals with spontaneous seizures showed increased inhibition in the dentate gyrus, a characteristic of the epileptic brain, with evidence of mossy fiber synaptic reorganization. Examination of circuit recruitment by c‐Jun immunohistochemistry showed activation restricted to the hippocampus in P21 animals subjected to PPS, although extensive activation of hippocampal and extrahippocampal structures was seen in pups subjected to PPS‐induced self‐sustaining SE at P35 or LiPC SE at P21. These results demonstrate that the appearance of epilepsy as a consequence of SE is influenced by the type of insult as well as by age‐dependent circuit recruitment. Ann Neurol 2000;48:580–589