Antiepileptic drugs and the developing brain.

Epilepsy is the most common neurological disorder in young humans. Antiepileptic drugs (AEDs) which are used to treat seizures in infants, children and pregnant women can cause cognitive impairment, microcephaly and birth defects. Ion channels, neurotransmitters and second messenger systems constitute molecular targets of AEDs. The same targets regulate brain processes essential both for propagation of seizures and for learning, memory and emotional behavior. Thus, AEDs can influence brain function and brain development in undesired ways. Here we review mechanisms of action of AEDs, examine clinical evidence for their adverse effects in the developing human brain, and present studies on cognitive and behavioral effects in animal models. Furthermore, we discuss mechanisms responsible for adverse effects of AEDs in the developing mammalian brain, including interference with cell proliferation and migration, axonal arborization, synaptogenesis, synaptic plasticity and physiological apoptotic cell death.

Acute and long-term proteome changes induced by oxidative stress in the developing brain.

The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration.

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.

Neurotransmitters and apoptosis in the developing brain.

In the immature mammalian brain during a period of rapid growth (brain growth spurt/synaptogenesis period), neuronal apoptosis can be triggered by the transient blockade of glutamate N-methyl-d-aspartate (NMDA) receptors, or the excessive activation of gamma-aminobutyric acid (GABA(A)) receptors. Apoptogenic agents include anesthetics (ketamine, nitrous oxide, isoflurane, propofol, halothane), anticonvulsants (benzodiazepines, barbiturates), and drugs of abuse (phencyclidine, ketamine, ethanol). In humans, the brain growth spurt period starts in the sixth month of pregnancy and extends to the third year after birth. Ethanol, which has both NMDA antagonist and GABA(A) agonist properties, is particularly effective in triggering widespread apoptotic neurodegeneration during this vulnerable period. Thus, maternal ingestion of ethanol during the third trimester of pregnancy can readily explain the dysmorphogenic changes in the fetal brain and consequent neurobehavioral disturbances that characterize the human fetal alcohol syndrome. In addition, there is basis for concern that agents used in pediatric and obstetrical medicine for purposes of sedation, anesthesia, and seizure management may cause apoptotic neuronal death in the developing human brain.

Apoptosis in the in vivo mammalian forebrain.

Apoptosis is a word originally introduced by Kerr, Wyllie, and colleagues for a cell death process they defined in terms of its ultrastructural appearance in nonneuronal cells from various tissues. There are very few studies providing detailed ultrastructural criteria for recognizing neuronal apoptosis in the in vivo mammalian brain. In the absence of such criteria, the Kerr/Wyllie description pertaining to nonneuronal cells has served as a reference standard. However, contemporary neurobiologists typically rely on cell culture models for studying neuronal apoptosis, and these models are rarely validated ultrastructurally; rather they are assumed to be appropriate models based on unvalidated biochemical tests for apoptosis. Relying on evidence generated in such cell culture models or on nonspecific cytochemical tests applied to brain tissue, many authors have recently suggested that an apoptotic mechanism may mediate neuronal death in a wide variety of human neurodegenerative diseases. Whether the cell death process in neurodegenerative diseases meets ultrastructural criteria for apoptosis has been given very little consideration. Recently, several methods have been described for triggering extensive apoptotic neurodegeneration in the developing in vivo mammalian brain. These methods include head trauma or treatment with several types of drugs (NMDA antagonists, GABAA agonists, or ethanol). We have performed an ultrastructural analysis of the neuronal cell death process triggered in the cerebral cortex and thalamus by these several methods and compared it with physiological cell death (PCD), a prototypic example of neuronal apoptosis that occurs naturally in the developing brain. Our findings, which are reviewed herein, demonstrate that the types and sequence of changes induced by each of the above methods are identical to those that characterize PCD. This confirms that each of these methods produces bona fide in vivo apoptotic neurodegeneration, and it signifies that our description of this neuronal apoptotic process, which differs in some respects from the Kerr/Wyllie description of nonneuronal apoptosis, can serve as a useful reference standard for recognizing the characteristic changes that in vivo neurons undergo when they are dying by an apoptotic mechanism.

Glutamate antagonists limit tumor growth.

Neuronal progenitors and tumor cells possess propensity to proliferate and to migrate. Glutamate regulates proliferation and migration of neurons during development, but it is not known whether it influences proliferation and migration of tumor cells. We demonstrate that glutamate antagonists inhibit proliferation of human tumor cells. Colon adenocarcinoma, astrocytoma, and breast and lung carcinoma cells were most sensitive to the antiproliferative effect of the N-methyl-d-aspartate antagonist dizocilpine, whereas breast and lung carcinoma, colon adenocarcinoma, and neuroblastoma cells responded most favorably to the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonist GYKI52466. The antiproliferative effect of glutamate antagonists was Ca(2+) dependent and resulted from decreased cell division and increased cell death. Morphological alterations induced by glutamate antagonists in tumor cells consisted of reduced membrane ruffling and pseudopodial protrusions. Furthermore, glutamate antagonists decreased motility and invasive growth of tumor cells. These findings suggest anticancer potential of glutamate antagonists.

Accumulation of the anandamide precursor and other N-acylethanolamine phospholipids in infant rat models of in vivo necrotic and apoptotic neuronal death.

It has been demonstrated that the endogenous cannabinoid receptor ligand, anandamide, and other N-acylethanolamines (NAEs), accumulate during neuronal injury in vitro, a process that may be linked to the neuroprotective effects of NAEs. The crucial step for generation of NAEs is the synthesis of the corresponding precursors, N-acylethanolamine phospholipids (NAPEs). However, it is unknown whether this key event for NAE formation is regulated differently in the context of insults causing necrotic or apoptotic neuronal death. To address this question, we monitored a range of cortical NAPE species in three infant rat models of in vivo neurodegeneration: (i) necrosis caused by intrastriatal injection of NMDA (25 nmol); (ii) apoptosis induced by systemic administration of the NMDA-receptor antagonist (+)MK-801 (3 x 0.5 mg/kg, i.p.); and (iii) apoptosis following focal necrosis triggered by concussive head trauma. A marked increase of all NAPE species was observed in both hemispheres 4 and 24 h after NMDA-induced injury, with a relatively larger increase in N-stearoyl-containing NAPE species. Thus, the percentage of the anandamide precursor fell from 1.1 to 0.5 mol %. In contrast, administration of (+)MK-801 did not alter cortical NAPE levels. Concussion head trauma resulted in a similar but less pronounced upregulation of NAPE levels at both 4 and 24 h as compared to NMDA injections. Increased levels of NAPE 24 h post-trauma possibly reflect that necrosis is still ongoing at this time point. Consequently, our data suggest that excitotoxic necrotic mechanisms of neurodegeneration, as opposed to apoptotic neurodegeneration, have a profound effect on in vivo NAE precursor homeostasis.

Glutamate signaling and the fetal alcohol syndrome.

It has been known for three decades that ethanol, the most widely abused drug in the world, has deleterious effects on the developing human brain, but progress has been slow in developing animal models that are optimal for studying this problem, and the underlying mechanisms have remained elusive. Recently, we have shown that during the synaptogenesis period, also known as the brain growth spurt period, ethanol has the potential to trigger widespread neuronal suicide (apoptosis), deleting many millions of neurons from the in vivo mammalian brain. It appears that ethanol triggers apoptotic neurodegeneration by a dual mechanism (blockade of NMDA glutamate receptors and excessive activation of GABA(A) receptors), in that ethanol has both NMDA antagonist and GABAmimetic properties; we have shown that other drugs which have either of these properties trigger apoptotic neurodegeneration in the developing brain. The brain growth spurt period in humans spans the last trimester of pregnancy and the first several years after birth. Thus, our findings provide a likely explanation for the reduced brain mass and neurobehavioral disturbances associated with the human fetal alcohol syndrome. Furthermore, since NMDA antagonist and GABAmimetic drugs are sometimes abused by pregnant women and also are used as anticonvulsants, sedatives, or anesthetics in pediatric medicine, our findings suggest the possibility that exposure of the developing brain to these various drugs either pre or postnatally could contribute to mental disability syndromes that have heretofore been attributed to unknown causes. In addition, the observation that ethanol and related drugs trigger massive neuronal apoptosis in the developing brain provides an unprecedented opportunity to study both neuropathological aspects and molecular mechanisms of apoptotic neurodegeneration in the in vivo mammalian brain.

Neuronal death enhanced by N-methyl-D-aspartate antagonists.

Glutamate promotes neuronal survival during brain development and destroys neurons after injuries in the mature brain. Glutamate antagonists are in human clinical trials aiming to demonstrate limitation of neuronal injury after head trauma, which consists of both rapid and slowly progressing neurodegeneration. Furthermore, glutamate antagonists are considered for neuroprotection in chronic neurodegenerative disorders with slowly progressing cell death only. Therefore, humans suffering from Huntington's disease, characterized by slowly progressing neurodegeneration of the basal ganglia, are subjected to trials with glutamate antagonists. Here we demonstrate that progressive neurodegeneration in the basal ganglia induced by the mitochondrial toxin 3-nitropropionate or in the hippocampus by traumatic brain injury is enhanced by N-methyl-d-aspartate antagonists but ameliorated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonists. These observations reveal that N-methyl-d-aspartate antagonists may increase neurodestruction in mature brain undergoing slowly progressing neurodegeneration, whereas blockade of the action of glutamate at alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors may be neuroprotective.

Mechanisms of neurodegeneration after paediatric brain injury.

Trauma to the developing brain constitutes an unexplored field. The few studies attempting to model and study paediatric head trauma, the leading cause of death and disability in the paediatric population, have revealed interesting aspects and potential targets for future research. One feature unique to the developing brain is overactivation by trauma of ongoing physiological programmed neuronal death (apoptosis). Understanding the underlying biochemical and molecular pathomechanisms may help set new pharmacotherapeutic targets for neuroprotection at an early age.

Environmental agents that have the potential to trigger massive apoptotic neurodegeneration in the developing brain.

We review recent findings pertaining to several environmental agents (ethanol, phencyclidine, ketamine, nitrous oxide, barbiturates, benzodiazepines, halothane, isoflurane, and propofol) that have the potential to delete large numbers of neurons from the developing brain by a newly discovered mechanism involving interference in the action of neurotransmitters [glutamate and gamma-amino butyric acid (GABA) at (italic)N(/italic)-methyl-d-aspartate (NMDA)] and GABA(subscript)A(/subscript) receptors during the synaptogenesis period, also known as the brain growth-spurt period. Transient interference (lasting >= 4 hr) in the activity of these transmitters during the synaptogenesis period (the last trimester of pregnancy and the first several years after birth in humans) causes millions of developing neurons to commit suicide (die by apoptosis). Many of these agents are drugs of abuse (ethanol is a prime example) to which the human fetal brain may be exposed during the third trimester by drug-abusing mothers. Ethanol triggers massive apoptotic neurodegeneration in the developing brain by interfering with both the NMDA and GABA(subscript)A(/subscript) receptor systems, and this can explain the reduced brain mass and lifelong neurobehavioral disturbances associated with intrauterine exposure of the human fetus to ethanol (fetal alcohol syndrome). Exposure of the immature brain in a medical treatment context is also of concern because many of these agents are drugs used frequently as sedatives, tranquilizers, anticonvulsants, or anesthetics in pediatric and/or obstetrical medicine. Because this is a newly discovered mechanism, further research will be required to fully ascertain the nature and degree of risk posed by exposure of the developing human brain to environmental agents that act by this mechanism.

Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome.

The deleterious effects of ethanol on the developing human brain are poorly understood. Here it is reported that ethanol, acting by a dual mechanism [blockade of N-methyl-D-aspartate (NMDA) glutamate receptors and excessive activation of GABA(A) receptors], triggers widespread apoptotic neurodegeneration in the developing rat forebrain. Vulnerability coincides with the period of synaptogenesis, which in humans extends from the sixth month of gestation to several years after birth. During this period, transient ethanol exposure can delete millions of neurons from the developing brain. This can explain the reduced brain mass and neurobehavioral disturbances associated with human fetal alcohol syndrome.

Ethanol-induced apoptotic neurodegeneration in the developing brain.

It has been known for three decades that ethanol, the most widely abused drug in the world, has deleterious effects on the developing human brain, but progress has been slow in developing animal models for studying this problem, and the underlying mechanisms have remained elusive. Recently, we have shown that during the synaptogenesis period, also known as the brain growth spurt period, ethanol has the potential to trigger massive neuronal suicide in the in vivo mammalian brain. The brain growth spurt period in humans spans the last trimester of pregnancy and first several years after birth. The NMDA antagonist and GABAmimetic properties of ethanol may be responsible for its apoptogenic action, in that other drugs with either NMDA antagonist or GABAmimetic actions also trigger apoptotic neurodegeneration in the developing brain. Our findings provide a likely explanation for the reduced brain mass and neurobehavioral disturbances associated with the human fetal alcohol syndrome. Furthermore, since NMDA antagonist and GABAmimetic drugs are sometimes abused by pregnant women and also are used as anticonvulsants, sedatives or anesthetics in pediatric medicine, our findings raise several complex drug safety issues. In addition, the observation that ethanol and several other drugs trigger massive neuronal apoptosis in the developing brain provides an unprecedented opportunity to study both neuropathological aspects and molecular mechanisms of apoptotic neurodegeneration in the in vivo mammalian brain.

Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain.

Age dependency of apoptotic neurodegeneration was studied in the developing rat brain after percussion head trauma. In 7-day-old rats, mechanical trauma, applied by means of a weight drop device, was shown to trigger widespread cell death in the hemisphere ipsilateral to the trauma site, which first appeared at 6 hours, peaked at 24 hours, and subsided by 5 days after trauma. Ultrastructurally, degenerating neurons displayed features consistent with apoptosis. A decrease of bcl-2 in conjunction with an increase of c-jun mRNA levels, which were evident at 1 hour after trauma and were accompanied by elevation of CPP 32-like proteolytic activity and oligonucleosomes in vulnerable brain regions, confirmed the apoptotic nature of this process. Severity of trauma-triggered apoptosis in the brains of 3- to 30-day-old rats was age dependent, was highest in 3- and 7-day-old animals, and demonstrated a subsequent rapid decline. Adjusting the mechanical force in accordance with age-specific brain weights revealed a similar vulnerability profile. Thus, apoptotic neurodegeneration contributes in an age-dependent fashion to neuropathological outcome after head trauma, with the immature brain being exceedingly vulnerable. These results help explain unfavorable outcomes of very young pediatric head trauma patients and imply that, in this group, an antiapoptotic regimen may constitute a successful neuroprotective approach.

Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain.

Much confusion has arisen recently over the question of whether excitotoxic neuronal degeneration can be considered an apoptotic phenomenon. Here, we addressed this question by using ultrastructural methods and DNA fragmentation analysis to compare a prototypic apoptotic in vivo central nervous system cell death process (physiologic cell death in the developing rat brain) with several central nervous system cell death processes in the in vivo infant rat brain that are generally considered excitotoxic (degeneration of hypothalamic neurons after subcutaneous administration of glutamate and acute neurodegeneration induced by hypoxia/ischemia or by concussive head trauma). We found by ultrastructural analysis that glutamate induces neurodegenerative changes in the hypothalamus that are identical to acute changes induced in the infant rat brain by either hypoxia/ischemia or head trauma, and that these changes are fundamentally different both in type and sequence from those associated with physiologic cell death (apoptosis). In addition, we show by ultrastructural analysis that concussive head trauma induces both excitotoxic and apoptotic neurodegeneration, the excitotoxic degeneration being very acute and localized to the impact site, and the apoptotic degeneration being delayed and occurring in regions distant from the impact site. Thus, in the head trauma model, excitotoxic and apoptotic degeneration can be distinguished not only by ultrastructural criteria but by their temporal and spatial patterns of expression. Whereas ultrastructural analysis provided an unambiguous means of distinguishing between excitotoxic and apoptotic neurodegeneration in each example analysed in this study, DNA fragmentation analysis (TUNEL staining or gel electrophoresis) was of no value because these tests were positive for both processes.

N-Methyl-D-aspartate antagonists and apoptotic cell death triggered by head trauma in developing rat brain.

Morbidity and mortality from head trauma is highest among children. No animal model mimicking traumatic brain injury in children has yet been established, and the mechanisms of neuronal degeneration after traumatic injury to the developing brain are not understood. In infant rats subjected to percussion head trauma, two types of brain damage could be characterized. The first type or primary damage evolved within 4 hr and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6-24 hr and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus and striatum. Secondary apoptotic damage was more severe than primary excitotoxic damage. Morphometric analysis demonstrated that the N-methyl-D-aspartate receptor antagonists 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonate and dizocilpine protected against primary excitotoxic damage but increased severity of secondary apoptotic damage. 2-Sulfo-alpha-phenyl-N-tert-butyl-nitrone, a free radical scavenger, did not affect primary excitotoxic damage but mitigated apoptotic damage. These observations demonstrate that apoptosis and not excitotoxicity determine neuropathologic outcome after traumatic injury to the developing brain. Whereas free radical scavengers may prove useful in therapy of head trauma in children, N-methyl-D-aspartate antagonists should be avoided because of their propensity to increase severity of apoptotic damage.

Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain.

Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.

Modeling pediatric head trauma: mechanisms of degeneration and potential strategies for neuroprotection.

We have developed a model for head trauma in infant rats in an attempt to study mechanisms of neurodegeneration in the developing brain and were able to morphologically characterize two distinct types of brain damage. The first type or primary damage evolved within 4 hrs after trauma and occurred by an excitotoxic mechanism. The second type or secondary damage evolved within 6-24 hrs and occurred by an apoptotic mechanism. Primary damage remained localized to the parietal cortex at the site of impact. Secondary damage affected distant sites such as the cingulate/retrosplenial cortex, subiculum, frontal cortex, thalamus, hippocampal dentate gyrus and striatum. Histological evidence of delayed cell death was preceded by decrease of bcl-2- in conjunction with increase of c-jun-mRNA-levels, already evident at 1 hr after trauma. Increase of CPP32-like activity and elevated concentrations of oligonucleosomes in affected brain regions represented additional findings to indicate that this secondary disseminated degenerative reaction is apoptotic in nature. At the age of 7 days, secondary apoptotic damage was more severe than primary excitotoxic damage, but its severity declined with increasing age. In 7-days-old rats, NMDA antagonists protected against primary excitotoxic damage but increased severity of secondary apoptotic damage whereas the free radical scavenger SPBN, the tumor necrosis factor (TNF) inhibitor pentoxifylline and the antioxidant N-acetylcystein mitigated apoptotic damage. These findings demonstrate that in the developing rat brain apoptosis and not excitotoxicity determines neuropathologic outcome following head trauma. Whereas radical scavengers and TNF-inhibitors may prove useful in treatment of pediatric head trauma, great caution should be applied in regards to the use of NMDA antagonists because of the inherent risk of apoptosis promotion.

Glutamate in neurologic diseases.

Excitotoxicity has been implicated as a mechanism of neuronal death in acute and chronic neurologic diseases. Cerebral ischemia, head and spinal cord injury, and prolonged seizure activity are associated with excessive release of glutamate into the extracellular space and subsequent neurotoxicity. Accumulating evidence suggests that impairment of intracellular energy metabolism increases neuronal vulnerability to glutamate which, even when present at physiologic concentrations, can damage neurons. This mechanism of slow excitotoxicity may be involved in neuronal death in chronic neurodegenerative diseases such as the mitochondrial encephalomyopathies, Huntington's disease, spinocerebellar degeneration syndromes, and motor neuron diseases. If so, glutamate antagonists in combination with agents that selectively inhibit the multiple steps downstream of the excitotoxic cascade or help improve intracellular energy metabolism may slow the neurodegenerative process and offer a therapeutic approach to treat these disorders.