Epileptogenesis and the Immature Brain

Summary: Epidemiological studies indicate that the incidence of seizures is highest early in life. This report discusses the experimental data derived from studies of focal epileptogenesis of the immature brain in tandem with ongoing maturational changes. During development, neurons have characteristic neurophysiological properties. Local interictal discharges are long in duration, lack a stereotypic morphology, and have limited fields. Yet the immature brain is very susceptible to the development of bilateral, although asynchronous, seizures and status epilepticus induced by amygdala kindling or by convul-sant drugs. This increased seizure susceptibility may be due to a functional immaturity of a substantia nigra, GABA-sensitive output system. The morbidity of convulsions occurring early in life may not be as grave as previously thought in terms of subsequent acquisition of "normal" developmental milestones. The propensity to develop recurrent convulsions in adulthood is not related to the severity of a single seizure in infancy. Although multiple severe seizures may predispose animals to the development of seizures later in life, this is not a unique feature of the immature brain, since it also occurs in the adult brain. Finally, there is evidence that the immature brain may respond to anticonvulsant drugs differently from its mature counterpart; these findings emphasize the need to develop new antiepileptic therapies that take into account the maturational state of the brain.     

Parenchymal Changes Related to Plasma Protein Extravasation in Experimental Seizures

To determine whether the transient opening of the blood-brain barrier (BBB) during epileptic seizures may lead to permanent neuronal changes, seizures of a few minutes' duration were induced by intravenous (i.v.) administration of 0.3 mg/kg bicuculline to conscious rats with indwelling catheters for blood pressure (BP) and blood gas monitoring. The rats were killed 5 min to 7 days later, and the distribution of endogenous plasma albumin, fibrinogen, and fibronectin in the brain was studied by immunohistochemistry. Parallel sections were scrutinized for evidence of light-microscopic structural changes in the tissue. Extensive multifocal extravasation of plasma proteins throughout the brain and brainstem was observed. The original clearly focal distribution became more diffuse with prolongation of the recovery time. In addition, the intensity of the immunoreactivity decreased, most likely due to drainage into the cerebrospinal fluid (CSF) in the ventricles and the subarachnoidal space of the extravasated proteins, but some antialbumin-positive material was still visible after 7 days. In areas with extravasation, many nerve cells, especially cerebellar Purkinje cells, became strongly positive for albumin. In some of these areas, neurons appeared to be irreversibly injured. Thus, considerable amounts of plasma proteins are extravasated even during short epileptic seizures, and albumin appear to remain in the tissue for a long time, especially in Purkinje cells. The Purkinje cell loss in chronic epilepsy may be caused partly by cumulative bouts of plasma extravasations.     

Immunocytochemistry of neuron specific enolase (NSE) in the rat brain after single and repeated epileptic seizures

The aim of this study was to investigate neuron-specific enolase (NSE) immunoreactivity of the different brain regions after pentylenetetrazol (PTZ)- induced epileptic seizures in rats. Light microscopic examinations provided evidences for changes of neuronal activity after single and repeated seizures. The number of NSE (+) cells was well correlated with Nissl staining. The results suggest that NSE immunoreactivity may be a valuable marker for determination of the number of metabolically active neurons in different brain regions after single and repeated experimental seizures.     

Functional integration of new hippocampal neurons following insults to the adult brain is determined by characteristics of pathological environment

We have previously shown that following severe brain insults, chronic inflammation induced by lipopolysaccharide (LPS) injection, and status epilepticus, new dentate granule cells exhibit changes of excitatory and inhibitory synaptic drive indicating that they may mitigate the abnormal brain function. Major inflammatory changes in the environment encountering the new neurons were a common feature of these insults. Here, we have asked how the morphology and electrophysiology of new neurons are affected by a comparably mild pathology: repetitive seizures causing hyperexcitability but not inflammation. Rats were subjected to rapid kindling, i.e., 40 rapidly recurring, electrically-induced seizures, and subsequently exposed to stimulus-evoked seizures twice weekly. New granule cells were labeled 1 week after the initial insult with a retroviral vector encoding green fluorescent protein. After 6–8 weeks, new neurons were analyzed using confocal microscopy and whole-cell patch-clamp recordings. The new neurons exposed to the pathological environment exhibited only subtle changes in their location, orientation, dendritic arborizations, and spine morphology. In contrast to the more severe insults, the new neurons exposed to rapid kindling and stimulus-evoked seizures exhibited enhanced afferent excitatory synaptic drive which could suggest that the cells that had developed in this environment contributed to hyperexcitability. However, the new neurons showed concomitant reduction of intrinsic excitability which may counteract the propagation of this excitability to the target cells. This study provides further evidence that following insults to the adult brain, the pattern of synaptic alterations at afferent inputs to newly generated neurons is dependent on the characteristics of the pathological environment.     

Multiple molecular penumbras after focal cerebral ischemia.

Though the ischemic penumbra has been classically described on the basis of blood flow and physiologic parameters, a variety of ischemic penumbras can be described in molecular terms. Apoptosis-related genes induced after focal ischemia may contribute to cell death in the core and the selective cell death adjacent to an infarct. The HSP70 heat shock protein is induced in glia at the edges of an infarct and in neurons often at some distance from the infarct. HSP70 proteins are induced in cells in response to denatured proteins that occur as a result of temporary energy failure. Hypoxia-inducible factor (HIF) is also induced after focal ischemia in regions that can extend beyond the HSP70 induction. The region of HIF induction is proposed to represent the areas of decreased cerebral blood flow and decreased oxygen delivery. Immediate early genes are induced in cortex, hippocampus, thalamus, and other brain regions. These distant changes in gene expression occur because of ischemia-induced spreading depression or depolarization and could contribute to plastic changes in brain after stroke.     

Unilateral cortical spreading depression is an early marker of audiogenic kindling in awake rats

Spreading depression (SD), a self-propagating wave of reversible cellular depolarization, is thought to play an important role in brain pathophysiology. SD and seizures are closely related events but little is known about involvement of SD in chronic epileptogenesis. Here we show that cortical SD is the first and highly reproducible manifestation of audiogenic kindling induced by repeated sound stimulation of WAG/Rij rats with genetic audiogenic and absence epilepsy. Repetition of sound-induced running seizures in freely moving rats led to an appearance and gradual intensification of post-running facial and forelimb clonic convulsions coupled with afterdischarge in the fronto-parietal cortex. Before the development of these traditional manifestations of audiogenic kindling, an unilateral cortical SD wave began to be triggered by audiogenic seizures. Once cortical SD appeared, it became a permanent component of subsequent seizures. SD was always recorded in the hemisphere ipsilateral to the running direction. Only at the late stages of audiogenic kindling SD developed bilaterally. To estimate the contribution of SD in postictal effects of audiogenic seizures, we compared cortical activity after seizures induced SD or not. It was found that only seizures with cortical SD were followed by postictal suppression of spontaneous spike-wave discharges displayed by WAG/Rij rats. The results show that (1) cortical SD is readily triggered by brief sensory-induced seizures in awake animals; (2) SD may be responsible for postictal changes in cortical activity; (3) unilateral initiation of SD suggests asymmetrical recruitment of the cortex into seizure network during audiogenic kindling.     

The pattern of 72-kDa heat shock protein-like immunoreactivity in the rat brain following flurothyl-induced status epilepticus

The inducible 72-kDa heat shock protein (HSP72) is a highly conserved stress protein that is expressed in CNS cells and may play a role in protection from neural injury. We used a monoclonal antibody to HSP72 and immunocytochemistry to localize HSP72 in the rat brain 24 h following either 30 or 60 min of flurothyl-induced status epilepticus. Sprague-Dawley rats were anesthetized with halothane, paralyzed, and ventilated, and remained normotensive and well oxygenated for the duration of the seizures. Seizure activity was quantified via analysis of the scalp EEG pattern. HSP72-like immunoreactivity (HSP72-LI) was induced in specific brain regions in a graded fashion that correlated, in part, with the duration and degree of seizure activity. Milder seizures produced HSP72-LI limited to layers 2 and 3 of frontoparietal cortex, dentate hilus cells, and CA3 pyramidal neurons. More extensive seizures led to HSP72-LI in layers 2, 3 and 5 of frontoparietal and visual cortex, dentate hilus cells, CA1 and CA3 pyramidal neurons, and certain thalamic and amygdaloid nuclei. These are similar to many, but not all, of the brain regions known to be injured with this model. No HSP72-LI was observed in sham-treated controls or flurothyl-treated animals whose seizures were controlled with pentobarbital. HSP72-LI thus localizes to certain regions of seizure-induced injury, and may provide a sensitive method of detecting neuronal 'stress' or injury relatively soon after status epilepticus. Whether or not HSP72 synthesis plays a protective role in the pathogenesis of seizures, or is only a marker for cell injury, remains to be determined.     

Pyruvate protects against kainate-induced epileptic brain damage in rats

Although the majority of epileptic seizures can be effectively controlled with antiepileptic drugs and/or surgery, a significant number progress to status epilepticus of sufficient duration to cause permanent brain damage. Combined treatment with antiepileptic drugs and neuroprotective agents, however, may help protect these individuals from permanent brain damage. Since toxicity induced by endogenous zinc contributes to epileptic brain injury, and since pyruvate is effective in reducing zinc-triggered neuronal death in cortical culture as well as ischemic neuronal death in vivo, we examined whether systemic pyruvate administration reduces seizure-induced brain damage. Na pyruvate (500 mg/kg) or osmolarity-matched saline (265 mg/kg NaCl, i.p.) were given to adult SD rats 30 or 150 min after 10 mg/kg kainite injection (i.p.), and there was no significant difference in the time course or severity of seizures between these groups. Zinc accumulation in neuronal cell bodies in the hippocampus, however, was much lower in the pyruvate than in the saline group. There was a close correlation between zinc accumulation and cell death, as assessed by acid-fuchsin and TUNEL staining. Pyruvate treatment markedly reduced neuronal death in the hippocampus, neocortex and thalamus. Pyruvate increased HSP-70 expression in hippocampal neurons. These results suggest that pyruvate, a natural glucose metabolite, may be useful as adjunct treatment in status epilepticus to reduce permanent brain damage.     

Consequences of epilepsy in the developing brain: implications for surgical management

The developing brain is highly susceptible to seizures, as demonstrated by both human and animal studies. Until recently, the brain has been considered to be relatively resistant to damage induced by seizures early in life. Accumulating evidence in animal models now suggests that early seizures can cause structural and physiologic changes in developing neural circuits that result in permanent alterations in the balance between neuronal excitation and inhibition, deficits in cognitive function, and increased susceptibility to additional seizures. The disruption of normal neuronal activity by seizures can affect multiple developmental processes, resulting in these long-lasting changes. These data should be considered in the clinical approach to children with intractable epilepsy and suggest that early intervention may avoid some of these long-term neurologic deficits.     

Cellular Mechanisms of Epilepsy and Potential New Treatment Strategies

Summary: Over the last 15 years, neurobiologists have begun to unravel the cellular mechanisms that underlie epileptiform activity. Such investigations have two main objectives: (I) to develop new methods for treating, "curing" or preventing epilepsy; and (2) to learn more about the normal functioning of the human brain at the cellular/ molecular and the neurological/psychological levels by analyzing abnormal brain functioning. The electroencephalogram (EEG) spike is a marker for the hyperexcitable cortex and arises in or near an area with a high epileptogenic potential. The depolarizing shift (DS) that underlies the interictal discharge (ID) appears to be generated by a combination of excitatory synaptic currents and intrinsic voltagedependent membrane currents. The hyperpolarization that follows the DS (post-DS HP) limits ID duration, determines ID frequency, and prevents ID deterioration into seizures. The disappearance of the post-DS HP in some models is related to the onset of seizures and the spread of epileptifonn activity. During the transition to seizures , the usually self-limited ID spreads in time and anatomical space. Several processes may intervene in the pathophysioogical dysfunction. These include enhancing GABA-mediated inhibition, dampening NMDA-mediated excitability, interfering with specific Ca²⁺ currents in central neurons, and perhaps stimulating "gating" pathways.     

Increased expression of gamma-aminobutyric acid transporter-1 in the forebrain of infant rats with corticotropin-releasing hormone-induced seizures but not in those with hyperthermia-induced seizures

High affinity, gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) influence the availability of GABA, the main inhibitory neurotransmitter in the brain. Recent studies suggest a crucial role for GATs in maintaining levels of synaptic GABA in normal as well as abnormal (i.e., epileptic) adult brain. However, the role of GATs during development and specifically changes in their expression in response to developmental seizures are unknown. The present study examined GAT-1-immunolabeling in infant rats with two types of developmental seizures, one induced by corticotropin-releasing hormone (CRH) lasting about 2 h and the other by hyperthermia (a model of febrile seizures) lasting only 20 min. The number of GAT-1-immunoreactive (ir) neurons was increased in several forebrain regions 24 h after induction of seizures by CRH as compared to the control group. Increased numbers of detectable GAT-1-ir cell bodies were found in the hippocampal formation including the dentate gyrus and CA1, and in the neocortex, piriform cortex and amygdala. In contrast, hyperthermia-induced seizures did not cause significant changes in the number of detectable GAT-1-ir somata. The increase in GAT-1-ir somata in the CRH model and not in the hyperthermia model may reflect the difference in the duration of seizures. The brain regions where this increase occurs correlate with the occurrence of argyrophyllic neurons in the CRH model.     

Basic Science

The hormone melatonin has been reported to exhibit antiepileptic properties in clinical trials. However, recent animal studies have demonstrated that melatonin can have opposite effects on brain function, depending on the dose and timing of melatonin administration. In other words, although high pharmacologic doses are able to decrease brain excitability and suppress seizures, smaller doses of melatonin (administered at night when melatonin levels in the brain are highest), similar in amount to what is produced by the brain, can actually increase the excitability of neurons, making them more susceptible to seizure activity. In this study, we used an animal model of epilepsy to study the effects of melatonin on seizure development. We made two important observations: (a) seizures induced by the drug pilocarpine occurred with a shorter latency at night (when brain melatonin levels are highest) than during the day, and (b) when small doses of drug that block melatonin receptors are injected directly into the hippocampus, an area of the brain important for the development and spread of seizures, then seizures during the night were delayed. Furthermore, this effect was reversed by a drug that blocks the activity of GABA, the major inhibitory neurotransmitter in the brain, suggesting that melatonin may decrease GABA-receptor function in the hippocampus. Although we did not study the effects of melatonin directly, our data suggest that endogenous melatonin may enhance brain excitability and contribute to the development of epileptic seizures. This process may be involved with certain forms of nocturnal epilepsy and may raise a caution for persons with epilepsy who take melatonin. Epilepsia 2005;46(4).     

Seizure-induced structural and functional changes in the rat hippocampal formation: comparison between brief seizures and status epilepticus

Prolonged seizures produce death of hippocampal neurons, which is thought to initiate epileptogenesis and cause a disruption of hippocampally mediated behaviors. This study aimed to evaluate behavioral and neuroanatomical changes induced by brief seizures and to compare them with changes induced by prolonged seizures. Adult rats were administered 6 brief seizures, elicited by electroshock (ECS). Prolonged seizures (status epilepticus, SE) were induced by pilocarpine. Two months later, the rats’ behavior was tested using the Morris water maze, passive avoidance and active avoidance tests. The number of neurons in the hippocampal formation was estimated using stereological methods. ECS seizures produced loss of neurons, ranging between 14% and 26%, in the dentate hilus, subiculum, presubiculum, parasubiculum, and entorhinal layers III and V/VI. However, the neuron loss caused by SE in the same structures, as well as in the hippocampal CA3 and CA1 fields, ranged between 34% and 50%. SE additionally killed many neurons in the dentate granular layer, postsubiculum and entorhinal layer II. ECS treatment caused mild impairments in spatial learning and passive avoidance, but it was not associated with spontaneous motor seizures. In contrast, SE produced a severe disruption of spatial learning, passive and active avoidance, and led to the development of spontaneous seizures. These data show that both prolonged seizure activity and brief seizures result in structural and functional alterations in the temporal lobe circuits, but those caused by prolonged seizures are considerably more severe. Hippocampal damage elicited by brief seizures does not necessarily lead to spontaneous motor seizures.     

Excitatory action of GABA on immature neurons is not due to absence of ketone bodies metabolites or other energy substrates

Brain slices incubated with glucose have provided most of our knowledge on cellular, synaptic, and network driven mechanisms. It has been recently suggested that γ-aminobutyric acid (GABA) excites neonatal neurons in conventional glucose-perfused slices but not when ketone bodies metabolites, pyruvate, and/or lactate are added, suggesting that the excitatory actions of GABA are due to energy deprivation when glucose is the sole energy source. In this article, we review the vast number of studies that show that slices are not energy deprived in glucose-containing medium, and that addition of other energy substrates at physiologic concentrations does not alter the excitatory actions of GABA on neonatal neurons. In contrast, lactate, like other weak acids, can produce an intracellular acidification that will cause a reduction of intracellular chloride and a shift of GABA actions. The effects of high concentrations of lactate, and particularly of pyruvate (4-5 mm), as used are relevant primarily to pathologic conditions; these concentrations not being found in the brain in normal "control" conditions. Slices in glucose-containing medium may not be ideal, but additional energy substrates neither correspond to physiologic conditions nor alter GABA actions. In keeping with extensive observations in a wide range of animal species and brain structures, GABA depolarizes immature neurons and the reduction of the intracellular concentration of chloride ([Cl(-)](i)) is a basic property of brain maturation that has been preserved throughout evolution. In addition, this developmental sequence has important clinical implications, notably concerning the higher incidence of seizures early in life and their long-lasting deleterious sequels. Immature neurons have difficulties exporting chloride that accumulates during seizures, leading to permanent increase of [Cl(-)](i) that converts the inhibitory actions of GABA to excitatory and hampers the efficacy of GABA-acting antiepileptic drugs.     

One of the hottest topics in epileptology: ABC proteins. Their inhibition may be the future for patients with intractable seizures

Abstract

One of the new topics in epileptology is the ABC proteins, which seem to control whether or not anti-epileptic drugs (AEDs) can come in contact with and affect the epileptogenic areas that cause seizures. The goal of this report is to simplify the concepts involved in these proteins and then to review the progress made in the field, especially of one protein called P-glycoprotein (P-gp). First, the ABC proteins are reviewed, mainly P-gp, which appears to alter drug permeability (like an extra blood–brain barrier). The possibility is discussed that changes in P-gp are the result of many seizures; are caused by the AEDs, or truly reflect pharmacoresistance. The different locations where these changes can be seen include the endothelial cells, glia and also neurons. The polymorphism of P-gp, called C3435T, probably has little functional significance and finally the importance of inhibitors of P-g to reverse pharmacoresistance is emphasized. Tariquidar (XR9576) is likely to be a good candidate that appears to inhibit these proteins and therefore to allow the AEDs to control the intractable seizures that may account for nearly 40% of our patients.     

Seizures in the developing brain result in a long-lasting decrease in GABAB inhibitory postsynaptic currents in the rat hippocampus

Whether seizures in the developing brain cause long-term changes in the mature brain has been debated. We tested the hypothesis that a model of early-life seizures, induced by systemic injection of a GABA_B receptor antagonist CGP56999A in immature rats, decreased GABA_B receptor-mediated inhibitory postsynaptic currents (IPSCs) in the hippocampus of adolescent rats. Whole-cell recordings were made in CA1 pyramidal cells and dentate gyrus (DG) granule cells in vitro, 30–45 days after the rats had seizures induced by CGP56999A (1–1.5 mg/kg i.p.) or control saline injection on postnatal day 15. GABA_B receptor-mediated IPSCs were reduced in DG neurons but not in CA1 neurons of early-life seizure rats as compared to controls. Additionally, hippocampal neurons of early-life seizure rats, as compared to those in control rats, showed a more depolarized resting membrane potential in both CA1 and DG, and a larger input resistance but reduced spike frequency adaptation in DG neurons. In conclusion, early-life seizures result in a long-lasting reduction in GABA_B receptor-mediated transmission in DG principal neurons and depolarization in CA1 and DG principal neurons. These alterations are expected to increase seizure susceptibility in the adult brain.     

The role of seizure-induced neurogenesis in epileptogenesis and brain repair

Data accumulated over the past four decades have led to the widespread recognition that neurogenesis, the birth of new neurons, persists in the hippocampal dentate gyrus and rostral forebrain subventricular zone (SVZ) of the adult mammalian brain. Neural precursor cells located more caudally in the forebrain SVZ are thought to also give rise to glia throughout life. The continued production of neurons and glia suggests that the mature brain maintains an even greater potential for plasticity after injury than was previously recognized. Underscoring this idea are recent findings that seizures induced by various experimental manipulations increase neurogenesis in the adult rodent dentate gyrus. Although neurogenesis and gliogenesis in persistent germinative zones are altered in adult rodent models of temporal lobe epilepsy (TLE), the effects of seizure-induced neurogenesis in the epileptic brain, in terms of either a pathological or reparative role, are only beginning to be explored. Emerging data suggest that altered neurogenesis in the epileptic dentate gyrus may be pathological and promote abnormal hyperexcitability. However, the presence of endogenous neural progenitors in other proliferative regions may offer potential strategies for the development of anti-epileptogenic or neuronal replacement therapies.     

Expression of heat shock proteins in Alzheimer's disease

In an investigation of heat shock proteins (HSPs) in the brains of Alzheimer's disease (AD) patients and cognitively intact control subjects, we found that 2 HSPs, termed "HSP72" and "GRP78," underwent major changes in expression in AD. HSP72, which was present at very low levels in control brains, increased dramatically in AD patients, and was localized exclusively in neuritic plaques and neurofibrillary tangles. We hypothesize that HSP72 is induced as an early response to the formation of abnormal proteins, perhaps targeting them for proteolysis. In contrast, GRP78 increased in AD only in neurons that remained cytologically normal, especially in the CA3 subfield of the hippocampus and the deep layers of the entorhinal cortex. The increased expression of GRP78 within successfully surviving neurons suggests that this protein may protect such cells from AD-specific damage.     

Spatiotemporal neuronal correlates of seizure generation in focal epilepsy

Purpose: Focal seizures are thought to reflect simultaneous activation of a large population of neurons within a discrete region of pathologic brain. Resective surgery targeting this focus is an effective treatment in carefully selected patients, but not all. Although in vivo recordings of single‐neuron (i.e., “unit”) activity in patients with epilepsy have a long history, no studies have examined long‐term firing rates leading into seizures and the spatial relationship of unit activity with respect to the seizure‐onset zone. Methods: Microelectrode arrays recorded action potentials from neurons in mesial temporal structures (often including contralateral mesial temporal structures) in seven patients with mesial temporal lobe epilepsy. Key Findings: Only 7.6% of microelectrode recordings showed increased firing rates before seizure onset and only 32.4% of microelectrodes showed any seizure‐related activity changes. Surprisingly, firing rates on the majority of microelectrodes (67.6%) did not change throughout the seizure, including some microelectrodes located within the seizure‐onset zone. Furthermore, changes in firing rate before and at seizure onset were observed on microelectrodes located outside the seizure‐onset zone and even in contralateral mesial temporal lobe. These early changes varied from seizure to seizure, demonstrating the heterogeneity of ensemble activity underlying the generation of focal seizures. Increased neuronal synchrony was primarily observed only following seizure onset. Significance: These results suggest that cellular correlates of seizure initiation and sustained ictal discharge in mesial temporal lobe epilepsy involve a small subset of the neurons within and outside the seizure‐onset zone. These results further suggest that the “epileptic ensemble or network” responsible for seizure generation are more complex and heterogeneous than previously thought and that future studies may find mechanistic insights and therapeutic treatments outside the clinical seizure‐onset zone.