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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Eur J Neurosci. Author manuscript; available in PMC Jun 1, 2013.
Published in final edited form as:
PMCID: PMC3383637
NIHMSID: NIHMS368642
Neonatal seizures: controversies and challenges in translating new therapies from the lab to the isolette
Kevin E. Chapman, MD, Yogendra H. Raol, PhD, and Amy Brooks-Kayal, MD
Corresponding Author: Amy Books-Kayal, MD, Professor of Pediatrics, Neurology and Pharmaceutical Sciences, University of Colorado Denver, Chief and Ponzio Family Chair, Pediatric Neurology, Children’s Hospital Colorado, 13123 East 16th Ave, B155, Aurora, CO 80045, Fax: 720-777-7285, amy.brooks-kayal/at/childrenscolorado.org
Abstract
Neonatal seizures have unique properties that have proved challenging for both clinicians and basic science researchers. Clinical therapies aimed at neonatal seizures have proven only partially effective and new therapies are slow to develop. This article will discuss neonatal seizures within the framework of the barriers that exist to the development of new therapies and the challenges inherent in bringing new therapies from the bench to the bedside. With the European Union and United States creating national collaborative project infrastructure, improved collaborative resources should advance clinical research on urgently needed new therapies for this disorder.
Keywords: Treatment, translational research, rodent models, epilepsy
Neonatal seizures constitute one of the most common neurologic issues in the newborn period and have engendered significant study and debate among neurologists. They are unique in many facets including their pathophysiology, treatment and outcome compared to seizures later in life. Much of this debate focuses on whether intensive treatment of neonatal seizures is necessary, as their impact on outcome remains unclear. In addition, the current treatments are often ineffective in controlling the seizures acutely and do not seem to impact the development of later epilepsy. This article will discuss the evidence supporting the deleterious effects of neonatal seizures in humans and animal models, future treatments, and the challenges in moving those therapies from the lab to clinical practice.
Neonatal seizures are common, with an incidence of 1.8–3.5 per 1000 live births (Saliba et al., 1999, Lanska et al., 1995). However, they can be difficult to identify clinically and are challenging to differentiate from a variety of normal, poorly coordinated, neonatal movements. Continuous EEG is currently the gold standard for identifying neonatal seizures, which may be subclinical >50% of the time (Scher et al., 1993). In a study comparing clinical identification of neonatal seizures by healthcare professionals with continuous EEG monitoring, only 27% of clinical seizures were correctly identified and 73% of presumed clinical seizure had no electrographic correlate - leading to overdiagnosis (Murray et al., 2008).
Neonatal seizures are most commonly associated with perinatal hypoxic-ischemic encephalopathy (Tekgul et al., 2006). This entity has been extensively studied and has a complicated pathophysiology that is mediated by excitatory amino acids, inflammatory cytokines, and free radical formation. Other common etiologies include vascular events, brain malformations, infections, and inborn errors of metabolism. Table 1 lists common etiologies associated with neonatal seizures and attempts that have been made to recreate these in rodent models for laboratory study. The etiology of the neonatal seizures is important to identify as it may have a significant effect on treatment (e.g. – pyridoxine-responsive seizures) and directly relates to outcome.
Table 1
Table 1
Etiology of neonatal seizures and available animal models
The incidence of seizures is highest in the first year of life (Hauser et al., 1993) and the risk of seizures is greatest in the neonatal period (Ronen et al., 1999, Saliba et al., 1999). Basic science research studies suggest that, compared to the mature brain, the developing brain is more excitable. The amount of chemoconvulsant required to induce seizures in immature animal is much lower than that required for induction of seizures in adult animals (Strafstrom et al., 1992). The enhanced excitability of the developing brain can be attributed to a variety of factors including early and exuberant development of excitatory neurotransmitter systems and comparatively delayed development of inhibition (for review see Holmes, 1997; Rakhade & Jensen, 2009). The increased neural activity associated with the enhanced excitation in the immature brain is essential for numerous activity-dependent developmental processes, but it also renders the developing brain more susceptible to seizures. Glutamate is the major excitatory neurotransmitter in the CNS that mediates its action via two types of receptors, metabotropic and ionotropic. The ionotropic receptors are further subdivided into N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA), and kainic acid (KA) receptors. During early postnatal development, NMDA and AMPA glutamate receptors are transiently overexpressed as compared to the mature brain and have a subunit composition that enhances excitability. Compared to the adult brain, for example, the immature brain has higher levels of NMDA receptor (NR) 2B proteins and lower levels of NR2A proteins (Monyer et al., 1994). The NMDA receptor that contains NR2B subunit, in place of NR2A subunit, has been shown to have a longer current decay time (Flint et al., 1997).
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the adult brain. However, due to developmental differences in the chloride gradient, GABA is excitatory in immature neurons (Ben-Ari et al., 1989). Potassium/chloride cotransporter 2 (KCC2), a cation chloride co-transporter channel important in extruding chloride out of the intercellular space, does not reach mature levels until after the neonatal period (Rivera et al., 1999). The chloride transporter sodium/potassium/chloride co-transporter 1 (NKCC1) predominates in the neonatal period and actively transports chloride into the cell (Plotkin et al., 1997; Dzhala et al., 2005). When GABAA receptors (a subtype of GABA receptors) are activated in immature neurons, the neuronal membrane is depolarized and there is a net excitatory effect, unlike the inhibitory effect of activating GABAA receptors in adult brain. Depolarizing GABA currents are critically important for normal activity-dependent developmental processes including neuronal proliferation, migration, targeting & synaptogenesis (LoTurco et al., 1995; Owens et al., 1996; Ben-Ari et al., 1997; Leinekugal et al., 1997). Moreover, in comparison to the adult brain, the immature brain has lower levels of GABAA receptors (Swann et al., 1989; Brooks-Kayal & Pritchett, 1993) and smaller GABA-mediated currents (Brooks-Kayal et al., 2001). Also, early in life, the subunit composition of GABAA receptors is different than that of the adult brain and this difference in subunits makes them less sensitive to benzodiazepine augmentation (Gibbs et al., 1996; Kapur & Macdonald 1999, Brooks-Kayal et al., 2001).
Results from animal research suggest that neonatal seizures may exacerbate hypoxia-ischemia induced brain injury (Dzhala et al., 2000; Wirrell et al., 2001; Björkman et al., 2010; but also see Towfighi et al., 1999). Björkman and colleagues (2010) observed that newborn piglets that were exposed to hypoxia and had seizures had greater brain injury compared to piglets without seizures. Neonatal seizures in rats have also been shown to cause long-term neurological problems. Adult rats that experienced flurothyl-induced recurrent neonatal generalized tonic-clonic seizures do not exhibit spontaneous seizures but have reduced seizure threshold (Isaeva et al., 2010). Multiple studies performed in various animal models suggest that rats develop cognitive and behavioral deficits in later life following early-life seizures (Lee et al., 2001; Sayin et al., 2004; Cornejo et al., 2008; Kleen et al., 2011). An elegant study by Cornejo and colleagues (2008) found that even a single episode of neonatal seizure in rats is sufficient to cause life-long alterations in working memory.
While basic science research would suggest that neonatal seizures can be harmful to the developing brain, there is a limited, but growing, literature supporting that this occurs in humans. Legido et al. (1991) found seizure frequency to be a strong predictor of the risk of developing a poor outcome and cerebral palsy in patients with asphyxia. However, this study did not use continuous EEG monitoring and likely underdiagnosed seizures. Similar results were found in a study evaluating neonates at risk for seizures, with the occurrence of electrographic seizures being associated with microcephaly and severe cerebral palsy (McBride et al., 2000). A 2002 study attempted to correlate the severity of seizures with MRI and 1H-MRS findings (Miller et al., 2002). The study found significant changes in the 1H-MRS spectra correlated to seizure severity suggesting that neonatal seizures in patients with hypoxia were associated with worsened brain injury. A study evaluating the utility of selective head cooling found that the presence of neonatal seizures at enrollment was associated with an unfavorable outcome at 18 months (Gluckman et al., 2005). A more recent study followed neonates with hypoxic-ischemic injury and clinical neonatal seizures to assess neurodevelopmental outcome at four years of age (Glass et al., 2009). They found that patients with HIE and neonatal seizures had worse motor and cognitive outcomes than those without seizures independent of the severity of their MRI findings. There is, of course, an inherent “chicken vs. egg” conundrum with studies of neonatal seizures in humans, and it is not possible to be certain that seizures “cause” additional brain injury and worsened outcome rather than being simply a marker of more severe underlying brain injury that may not be detectable by history, exam or current neuro-imaging techniques. However, with this caveat and in combination with studies in rodent models, the preponderance of the evidence seems to support a contributory role of neonatal seizures to brain injury and subsequent neurological disability.
What are the changes in the brain caused by neonatal seizures that may contribute to long-term neurological disability in certain patients later in life? In human patients, it is very difficult to parse out the effects of the etiology of the seizures and the effects of treatment from the effects of seizures on the developing brain. Studies conducted in animal models have identified cell death (Kadam & Dudek, 2007), aberrant synaptic connections (Grigonis & Murphy, 1994; Sogawa et al., 2001; Kadam & Dudek, 2007; Xiu-Yu et al., 2007; Rakhade et al., 2011), increases in thickness of prefrontal cortex (Kleen et al., 2011) and changes in rate of neurogenesis (McCabe et al., 2001; Liu et al. 2003; Xiu-Yu et al., 2007; for review see Porter, 2008) following neonatal seizures. In addition to structural modifications, chronic suppression of inhibitory activity (Isaeva et al., 2009), a long-term increase in excitatory activity (Isaeva et al., 2010) and modifications in subunit composition of GABA (Zhang et al., 2004a; Laurén et al, 2005) and glutamate receptors (Zhang et al., 2004b; Silva et al., 2005; Swann et al., 2007; Cornejo et al., 2007) have been observed after the occurrence of neonatal seizures (for review see Holopainen, 2008). Persistent changes in inhibitory and excitatory pathways may explain some of the neurologic sequelae, such as increased seizure susceptibility and memory impairment, observed in later life following neonatal seizures. Cornejo and colleagues (2007) found impairment in working memory associated with alterations in synaptic plasticity, increase in intracellular levels of GluR1 subunit (reduced expression at the membrane) and reduced levels of NR2A subunit. However, currently these associations remain correlative and direct evidence that connects neonatal seizures to adverse neurological outcome in later life remains elusive.
Identification of neonatal seizures
One of the limitations for clinical research in neonatal seizures is accurate identification of seizure activity. Amplitude-integrated EEG (aEEG) has become more widely available due to its ease of application (fewer recording channels and can be applied by bedside nursing) and lower cost compared to traditional EEG. Many recent studies have attempted to correlate the seizures noted on aEEG with outcome to determine if the seizures are independently associated with a worse outcome. This technique likely allows for better screening of at-risk infants, though has well documented limitations with lower seizure detection rates for brief, low amplitude, or focal seizures (Toet et al., 2002, review by Tao & Mathur, 2011). In a retrospective study of patients who underwent aEEG and were found to have neonatal status epilepticus (SE), the subgroup of patients with HIE and a poor outcome had a longer duration of status epilepticus (van Rooij et al., 2007). Interestingly, this did not hold true statistically for other causes supporting the idea that the HIE subgroup may be particularly at risk for injury from neonatal SE. A recent randomized study evaluated the treatment of clinical and subclinical seizures identified by aEEG in patients with HIE (van Rooij et al., 2010). The study found that there was a significant correlation between the severity of brain injury on MRI and the duration of seizure activity. Treatment of subclinical seizures shortened the total duration of seizure patterns and reduced brain injury, suggesting that patients at risk for HIE should be monitored electrographically to identify and treat these subclinical seizures.
Lack or paucity of good treatment options
Effective treatment of neonatal seizures has proven challenging with studies suggesting that traditional therapies are only modestly effective. In a study comparing the efficacy of phenobarbital to phenytoin, seizure control was achieved in only about 45% of the patients following administration of the first medication (Painter et al., 1999). The patients were then given the alternate medication - increasing seizure control to only about 60%. This study highlighted that 40% of patients continued to have seizures despite treatment with two conventional antiepileptic medications. Benzodiazepines have been advocated for the treatment of neonatal seizures and may be effective in refractory patients. In one study, midazolam was effective in controlling electrographic seizures in patients who had failed first-line therapy with phenobarbital or phenytoin (Castro Conde et al., 2005). Lidocaine has also proven effective for neonatal seizures that have failed to respond to traditional anticonvulsant medications; however, concerns for cardiac toxicity has limited its widespread use (Malingré et al., 2006). Topiramate (Glass et al., 2011) and levetiracetam (Abend et al., 2011) have shown some efficacy in preliminary retrospective studies. Table 2 lists neonatal seizure treatments and their presumed therapeutic targets. All of the currently available antiepileptic drugs, including phenobarbital, have been developed using adult animal models and tested clinically in adult patients. However, there are significant anatomical, electrophysiological and neurochemical differences between the developing and mature brain. Therefore, immature brain may respond very differently than the adult brain to both injury and treatment. As described above, in comparison to the adult brain, GABA as an inhibitory system in early life is less well developed and even has an excitatory activity that may explain the poor efficiency of phenobarbital and diazepam (which act by augmenting GABAA receptor activity) to treat neonatal seizures. Another important factor to be considered while treating neonatal seizures (in fact any disease), that is often neglected, is the gender of the patient. A recent study demonstrated that the hyperpolarizing reversal potential of GABAergic postsynaptic currents appear earlier in female than in male rat hippocampus and neonatal seizures had very different immediate effects on the GABAergic system in animals of different gender (Galanopoulou, 2008). These differences may affect the efficacy of GABAergic drugs as well as later neurologic outcomes.
Table 2
Table 2
Treatment of neonatal seizures
Risk of treatment
A major consideration for physicians treating neonates with seizures is the potential for deleterious effects of seizure treatment on the developing brain. Several studies have shown that in utero exposure to certain antiepileptic drugs (AEDs) can increase the risk of cognitive dysfunction later in life (Meador et al., 2009, for review see Bromley et al., 2009). In children with seizures treated with AEDs, the effects of AED treatment on cognition can be difficult or impossible to differentiate from those of the seizures and/or underlying cause of the epilepsy; although, in the well-known study of children with febrile seizures randomized to placebo or phenobarbital, Farwell and colleagues demonstrated a persistent decrease in the mean IQ of the phenobarbital treated group (Farwell et al., 1990). No such placebo controlled randomized studies in humans exist that examine potential cognitive effects of AED treatment of neonates, however based on animal research there is potential risk. In rodent models, there is evidence that neonatal AED exposure, especially with older drugs such as phenobarbital, phenytoin, and valproic acid, alters a number of activity-dependent developmental processes, including neuronal gene expression, migration, differentiation and survival (for review see Marsh et al., 2006).
NKCC1 transporter inhibitors
Bumetanide, a loop diuretic, may prove to be a valuable adjunctive therapy for neonatal seizures. As described above, due to developmental differences in chloride transporter expression (higher NKCC1 levels and lower KCC2 levels in immature brain), during early development GABA has excitatory activity (Ben-Ari et al., 1989; Plotkin et al., 1997; Rivera et al., 1999; Dzhala et al., 2005). Bumetanide inhibits the NKCC1 transporter, altering the chloride gradient such that GABA channel opening is more hyperpolarizing and possibly allowing GABAergic medications to be more effective. There are currently two multicenter clinical trials evaluating the efficacy of bumetanide in neonates with hypoxic-ischemic encephalopathy.
Hypothermia
Therapeutic hypothermia has proven effective in improving outcomes in moderate to severe hypoxic-ischemic encephalopathy (Shankaran et al., 2005, Gluckman et al., 2005). Brain cooling likely modulates multiple neurotoxic processes including decreased cerebral metabolism, ion pump dysfunction, formation of cytotoxic edema, free radical formation, and neuroinflammation (Polderman, 2009). Limited case series suggest that it may be effective for status epilepticus in children and adults, but evidence for the efficacy of hypothermia in the treatment of seizures in neonates is limited. (Rossetti, 2011). A recent study of neonates undergoing therapeutic hypothermia found that 65% had electrographic seizures during or immediately after cooling, suggesting that hypothermia may have limited impact on the incidence of seizures (Wusthoff et al., 2011). Harbert et al. reviewed patients with focal neonatal stroke who underwent therapeutic hypothermia for neonatal encephalopathy and compared them with subjects with neonatal stroke who did not receive hypothermia. They found that none of the five patients with focal stroke and hypothermia developed seizures, while 70% of those who did not receive hypothermia developed seizures (Harbert et al., 2011). While therapeutic hypothermia may have a positive impact on hypoxic-ischemic encephalopathy, its utility as a treatment option for neonatal seizures remains questionable.
AMPA receptor modulators
AMPA receptors are constructed from GluR1-4 subunits. Different combinations of the subunit produce AMPA receptors with distinct function. For example, the presence of GluR2 subunits reduces permeability of AMPA receptors to calcium ions (Bochet et al., 1994). The immature rodent and human brain have a higher number of AMPA receptors that lack GluR2 subunits and gate greater amounts of calcium (for review see Jensen, 2002, Talos et al., 2006a,b). Post-translational modifications, such as alternative splicing of the AMPA receptor subunits, also affects AMPA receptor functional properties. Alternative splicing of AMPA receptor subunits generates flip and flop isoforms of the subunits (Rogawski et al., 1999). The AMPA receptor that has subunits with the flip configuration desensitizes at much slower rate than the AMPA receptors whose subunits are in the flop configuration (Rogawski et al., 1999). The immature brain has higher flip/flop ratio than the adult brain, which might contribute to the hyperexcitability of the developing brain (Monyer et al., 1991; for review see Dingledine et al., 1999). In fact, in a neonatal rat model of hypoxia-ischemia, Jensen and colleagues (1995) observed that the AMPA receptor antagonist NBQX effectively blocked acute seizures, whereas GABA agonists such as phenobarbital were ineffective in stopping seizures. Similarly topiramate, which has been shown to block AMPA receptor activity, was found to be effective in suppressing hypoxia-ischemia induced neonatal seizures in rats (Koh & Jensen, 2001). In a more recent study, pretreatment of neonatal rats with talampanel, a noncompetitive antagonist of AMPA receptors, was found to be effective in preventing the development of seizures during hypoxia exposure (Aujla et al., 2009). These studies suggest that AMPA receptor antagonists may prove an effective treatment for neonatal seizures.
Potassium channel openers
Potassium channels play a uniquely important role in controlling excitability in the developing brain because of the lower levels of GABAergic inhibition. Mutations in genes encoding KCNQ2 and KCNQ3 subunits of voltage gated potassium channels cause benign familial neonatal convulsions (BFNC), a genetic epilepsy syndrome (Singh et al., 2003). One of the interesting characteristics of BFNC is that the seizures begin in the first week of life and usually spontaneously remit after a few weeks or months. This suggests that the potassium channels play a particularly critical role in controlling hyperexcitability during the neonatal period and early infancy. A view further supported by a study in rodents that demonstrated that the blockade of KCNQ2/3 channel activity in early development results in development of severe epilepsy; whereas, blockade of KCNQ2/3 channel activity during adulthood results in a much milder phenotype (Peters et al., 2005). These observations suggest that a potassium channel opener can be a highly effective way to enhance inhibition and treat neonatal seizures. A recent study showed excellent efficacy of flupirtine, a potassium channel opener, in treating neonatal seizures in rats (Raol et al., 2009). Flupirtine, unlike diazepam and phenobarbital, completely blocked neonatal seizures induced by chemoconvulsants and when administered 15 minutes after rats had developed continuous seizures effectively stopped electrographic and behavioral seizures.
Bringing a new effective therapeutic treatment to market is an expensive and prolonged process with multiple challenges. The ideal goal is to have a firm understanding of the pathophysiologic processes leading to disease and tailor treatments to modify or block these processes. While our understanding of neonatal seizures continues to grow, translational research has proven difficult. Many of the features that make the neonatal brain uniquely susceptible to seizures, such as differences in the chloride gradient that diminish the inhibitory effects of GABA and exuberant expression of glutamate receptors that enhance excitability, are critical for driving normal activity-dependent developmental processes. Thus, treatments that target these mechanisms to reduce seizures have the potential to produce deleterious effects on normal neurocognitive development that must be carefully monitored (and somehow differentiated from those of the underlying brain injury and seizures themselves). Essential to our ability to establish therapies for neonatal seizures that are both safe and effective are improved animal models. Current models of neonatal seizures are overly simplistic and largely unrepresentative of the typical etiologies of seizures in human neonates. Further, many of the most concerning potential outcomes from neonatal seizures, such as neurobehavioral and neurocognitive abnormalities of language and executive function, are poorly assessed in most rodent models. In order to improve translational research, better models and better methods of assessing cognitive and behavioral outcomes after neonatal seizures need to be identified.
Lack of good animal models
Since the discovery of the anticonvulsant activity of phenytoin in cats (Putnam & Meritt, 1937), experimental models of seizures and epilepsy have played a tremendously important role in improving our understanding of the disease process and discovering newer treatment options. A good animal model that replicates all of the important aspects of the disease it is modeling is the most important tool required in the fight against disease. A good animal model of an epilepsy disorder will accurately replicate the etiology of the disorder, the age of onset of the disorder, the seizure phenotype, the EEG characteristics and the long-term consequences of the disorder (Sarkisian, 2001; Stafstrom et al., 2006). As mentioned earlier in this review and as pointed out in the report from the National Institute of Health (NIH) workshop about models of epilepsy and epileptogenesis, the existing programs for antiepileptic drug discovery have aimed at identifying therapies for the adult, rather than the pediatric, population (Stables et al., 2002). The developing brain is not a smaller version of the adult brain; therefore, to identify the most effective therapeutic intervention strategy, it is imperative to target age-specific mechanisms and test new therapies in age-specific disease models. In recent years, the NIH and the epilepsy research community has put a great emphasis on the development of model systems specific for pediatric epilepsies (Stables et al., 2002; Stafstrom et al., 2006). As a result, a significant increase in the research activity directed towards the development of newer models for childhood epilepsies has occurred, which is evident by three newly proposed animal models of infantile spasm in as many years (Marsh et al., 2009; Price et al., 2009; Scantlebury et al., 2010). As shown in Table 1, there are some animal models that replicate the etiology of human neonatal seizures, however many (such as common chemoconvulsant models such as kainate and flurothyl) do not, but rather model general phenomena of excitotoxicity. In addition, most of the animal models of neonatal seizures have not been validated in terms of the presence of electrographic seizures (which often do not have predictable behavioral correlates) and the development of epilepsy in later life has not been established in many of these models. It is extremely challenging to characterize behavioral or electrographic seizures in newborn animals due to their smaller size (mouse and rat pup) and due to the fact that the newborns cannot be kept separate from the mother for a very long time. Moreover, almost all of the available animal models are created using animals that are normal and do not have any brain pathology, which is in contrast to what is observed in the majority of human patients (for review see Lombroso, 2007). The other challenge in developing a good neonatal seizure model is identifying an age in the animal that accurately correlates with the human neonate (for review see Avishai-Eliner et al., 2002; Watson et al., 2006). Studies of synaptogenesis, neuroanatomy, metabolism and neurotransmitters receptors expression suggest that the first year of human life is roughly equivalent to 7–14 days of life in the rat. However, the inter-species age correlation may vary depending on the specific developmental factor considered for comparison. A comparison of total brain weight gain as a percentage of adult weight suggest that a 5 to 7 day old rat is equivalent to a human newborn (Dobbing & Sands, 1979), whereas, a comparison of development of hippocampus between human and rat suggest that the first week of life in rats might be comparable to the third trimester of gestation in a human (Avishai-Eliner et al., 2002). Cortical glutamate decarboxylase activity in 7.4 to 9 days old rat is comparable to a 40 week post-conceptional human (Romijn et al., 1991), whereas, comparison of electrical activity recorded using aEEG suggest that 10-day old Wistar rat brain is equivalent to a new born (Tucker et al., 2009). Further, the developmental changes in the brain often vary between two strains of the same species. For example, the developmental changes in AMPA receptor subunit expression in the cortex occurs 2–3 days earlier in Sprague-Dawley rats than in the Long-Evans rats, which could be due to differences in the gestation period between the two strains (Talos et al., 2006b).
Preclinical studies
Drug development involves three major steps: (1) basic science research, (2) preclinical studies and, (3) clinical trials. Basic science research is required to discover a physiological target that can be manipulated to modify the disease. Preclinical studies help with identification of a compound that can modulate the target. Preclinical studies also determine pharmacological and toxic properties of the test compound in animal models. Translating treatments from the rodent to the human can be particularly challenging as there are significant differences between the species that may affect the utility of the treatment, such as differences in metabolism or toxicity. Translation of neonatal treatments can be especially challenging given the pharmacodynamic differences between adults and neonates (for review see Stephenson, 2005). Funding is, of course, always a substantial challenge. In US, the main source of funding for basic science and preclinical research is the NIH. Private foundations, such as Citizens United for Research in Epilepsy (CURE) and other state and federal programs, also provide vital support for the drug discovery research. For preclinical development and early clinical trials, NIH provides resources such as the Rapid Access to Interventional Development (RAID) pilot program, Small Business Innovative Research (SBIR) program, Small Business Technology Transfer (STTR) program, Rapid Access to Preventive Intervention Development (RAPID) program, and UO1 grant program. However, in recent years research funding has become more scarce due to reductions in the NIH budget. Moreover, because of the economic downturn, private foundations have seen reductions in donations. Limitations on the amount of funding affects the development of new drugs in multiple ways, including the ability to accurately translate rodent study data for human use. For example, calculating an effective dose of a drug that does not have any side effects in human based on the data from rodents is not only complicated, it can also be inaccurate (Reagan-Shaw et al., 2008). If a drug is found to be effective in treating a disease in a rodent model, it would be ideal to test its effects in a primate model before its use in the humans. However, primate research is very expensive and involves complex ethical questions.
Clinical trials
Clinical trials in children and neonates are complicated by ethical, physiologic, pharmacologic, neurodevelopental and economic concerns (Kern, 2009). Consent, beneficence, confidentiality and equipoise are important considerations in the design of all trials, but may be particularly challenging in neonates who are often critically ill. The varied physiologic and pharmacologic responses and interactions of medications in neonates are difficult to extrapolate from adult studies and critical phase I and II trials are often lacking, making clinical trials difficult to formulate safely. There are economic concerns for drug manufacturers when medications have a limited market – particularly in small affected populations or rare diseases and with short treatment periods (most neonates are treated for seizures for a period of only a few weeks). Companies often cite the limited market for some of these drugs and the relatively poor return on investment in performing trials in children. This process of bringing a drug to market typically takes between 8 and 12 years and is estimated to cost $403 million of dollars per drug (DiMasi et al., 2003). Typically only one of five thousand to ten thousand compounds actually becomes approved for marketing. A recent study found that only 8% of CNS drugs that entered clinical testing were successful in achieving clinical approval, the lowest of all therapeutic classes (DiMasi et al., 2010). There has been an increasing complaint from drug manufacturers that there are limited affected patients available for studies in the United States and Europe. Sponsors have found it challenging to find sufficient US investigators and subjects and have been conducting more trials overseas in central Europe and Asia (Krall et al., 2011). This can be particularly problematic for studies in children and neonates for diseases that have a low prevalence in the population. In the absence of adequate support from the pharmaceutical industry, funding from governmental agencies such as NIH will be critical. Due to their relatively low prevalence, adequate clinical trials for neonatal seizures will require involvement of multiple sites and a long follow-up period of 5–10 or more years will be needed to adequately assess neurodevelopmental outcome, making such studies both expensive and logistically complex.
Childhood cancer trials have been enormously successful at lowering mortality rates for relatively rare childhood cancers through a network of national and multinational research groups. Neonatal seizures, while relatively common, may benefit from a similar framework for amassing patients and funneling promising projects through a series of academic centers with the expertise to conduct high-quality research in a collaborative way. The European Union and United States have created programs with the goal of streamlining research and drug approval. In the European Union, the Innovative Medicine Initiative (IMI) and the European Seventh Framework Programme (FP7) are building collaborative research efforts between academic centers as well as commercial biomedical partners. For example, The Treatment of Neonatal seizures with Medications Off-patent: evaluation of efficacy and safety of bumetanide (NEMO) trial is funded through the FP7 program. This study will evaluate the efficacy of bumetanide in neonates following hypoxic-ischemic injury. A similar trial is underway in the United States, with both of these trials striving toward a successful transition from bench to bedside with government sponsored support.
In the United States, the NIH created the Clinical and Translational Science Awards (CTSA) to partner 60 academic centers and the private sector to facilitate utilization of scalable assets for investigator use. The NINDS has recently introduced the NeuroNEXT program to provide a standardized and accessible infrastructure to support Phase II trials in pediatric and adult neurologic diseases. This program will centralize the institutional review process, provide statistical support and assist researchers in trial design and applying for funding. This will hopefully bridge some of the gap between basic science and clinical research. Trials looking to enroll patients with rare diseases will be able to utilize the multicenter approach to identify and enroll patients.
Neonatal seizures are heterogeneous and complex and it will require improved understanding of basic mechanisms, improved disease models, and better strategies for performing clinical trials to successfully bring effective therapies to market for clinical use. With new programs in Europe and the United States addressing some of these concerns, there will hopefully be an improved transition of new therapies from the basic science lab to the isolette.
Acknowledgments
supported by funding from CURE (YHR) and NICHD RO1HD065534 (YHR)
  • Abend NS, Gutierrez-Colina AM, Monk HM, Dlugos DJ, Clancy RR. Levetiracetam for treatment of neonatal seizures. J Child Neurol. 2011;26:465–470. [PMC free article] [PubMed]
  • Alles YC, Greggio S, Alles RM, Azevedo PN, Xavier LL, DaCosta JC. A novel preclinical rodent model of collagenase-induced germinal matrix/intraventricular hemorrhage. Brain Res. 2010;1356:130–138. [PubMed]
  • Aujla PK, Fetell MR, Jensen FE. Talampanel suppresses the acute and chronic effects of seizures in a rodent neonatal seizure model. Epilepsia. 2009;50:694–701. [PMC free article] [PubMed]
  • Avishai-Eliner S, Brunson KL, Sandman CA, Baram TZ. Stressed-out, or in (utero)? Trends Neurosci. 2002;25:518–524. [PMC free article] [PubMed]
  • Backman SA, Stambolic V, Suzuki A, Haight J, Elia A, Pretorius J, Tsao MS, Shannon P, Bolon B, Ivy GO, Mak TW. Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease. Nat Genet. 2001;29:396–403. [PubMed]
  • Balasubramaniam J, Del Bigio MR. Animal models of germinal matrix hemorrhage. J Child Neurol. 2006;21:365–371. [PubMed]
  • Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa JL. Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol. 1989;416:303–25. [PubMed]
  • Ben-Ari Y, Khazipov R, Leinekugel X, Caillard O, Gaiarsa JL. GABAA, NMDA and AMPA receptors: a developmentally regulated ‘ménage à trois’ Trends Neurosci. 1997;20:523–529. [PubMed]
  • Björkman ST, Miller SM, Rose SE, Burke C, Colditz PB. Seizures are associated with brain injury severity in a neonatal model of hypoxia-ischemia. Neuroscience. 2010;166:157–67. [PubMed]
  • Bochet P, Audiant E, Lambolez B, Crepel F, Rossier J, Lino M, Tsuzuki K, Ozawa S. Subunit composition at the single-cell level explains functional properties of a glutamate gated channel. Neuron. 1994;12:383–388. [PubMed]
  • Bonthius DJ, Perlman S. Congenital viral infections of the brain: lessons learned from lymphocytic choriomeningitis virus in the neonatal rat. PLoS Pathog. 2007;3:e149. [PMC free article] [PubMed]
  • Bromley RL, Baker GA, Meador KJ. Cognitive abilities and behavior of children exposed to antiepileptic drugs in utero. Curr Opin Neurol. 2009;22:162–166. [PMC free article] [PubMed]
  • Brooks-Kayal A, Pritchett D. Developmental changes in human GABAA receptor subunit composition. Ann Neurol. 1993;34:687–693. [PubMed]
  • Brooks-Kayal A, Shumate M, Jin H, Rikhter T, Kelly M, Coulter D. GABA(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development. J Neurochem. 2001;77:1266–1278. [PubMed]
  • Castro Conde JR, Hernandez Borgez AA, Domenech Martinez E, Gonzalez Campo C, Perera Soler R. Midazolam in neonatal seizures with no response to phenobarbital. Neurology. 2005;64:876–879. [PubMed]
  • Cherian SS, Love S, Silver IA, Porter HJ, Whitelaw AG, Thoresen M. Posthemorrhagic ventricular dilation in the neonate: development and characterization of a rat model. J Neuropathol Exp Neurol. 2003;62:292–303. [PubMed]
  • Comi AM, Johnston MV, Wilson MA. Immature mouse unilateral carotid ligation model of stroke. J Child Neurol. 2005;20:980–3. [PubMed]
  • Cornejo BJ, Mesches MH, Coultrap S, Browning MD, Benke TA. A single episode of neonatal seizures permanently alters glutamatergic synapses. Ann Neurol. 2007;61:411–426. [PubMed]
  • Cornejo BJ, Mesches MH, Benke TA. A single early-life seizure impairs short-term memory but does not alter spatial learning, recognition memory, or anxiety. Epilepsy Behav. 2008;13:585–92. [PMC free article] [PubMed]
  • Crino PB. Focal brain malformations: seizures, signaling, sequencing. Epilepsia. 2009;50(Supp 9):3–8. [PubMed]
  • Dingledine R, Borges K, Bowie D, Traynelis SF. The glutamate receptor ion channels. Pharmacol Rev. 1999;51:7–61. [PubMed]
  • DiMasi JA, Hansen RW, Grabowski HG. The price of innovation: new estimates of drug development costs. J Health Econ. 2003;22:151–185. [PubMed]
  • DiMasi JA, Feldman L, Seckler A, Wilson A. Trends in risk associated with new drug development: success rates for investigational drugs. Clin Pharmacol Ther. 2010;87:272–277. [PubMed]
  • Dobbing J, Sands J. Comparative aspects of the brain growth spurt. Early Hum Dev. 1979;3:79–83. [PubMed]
  • Dzhala V, Ben-Ari Y, Khazipov R. Seizures accelerate anoxia-induced neuronal death in the neonatal rat hippocampus. Ann Neurol. 2000;48:632–640. [PubMed]
  • Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ. NKCC1 transporter facilitates seizures in the developing brain. Nature Medicine. 2005;11:1205–1213. [PubMed]
  • Eyles D, Brown J, Mackay-Sim A, McGrath J, Feron F. Vitamin D3 and brain development. Neuroscience. 2003;118:641–653. [PubMed]
  • Farwell JR, Lee YJ, Hirtz DG, Sulzbacher SI, Ellenberg JH, Nelson KB. Phenobarbital for febrile seizures – effects on intelligence and on seizure recurrence. N Engl J Med. 1990;322:364–369. [PubMed]
  • Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H. NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J Neurosci. 1997;17:2469–2476. [PubMed]
  • Galanopoulou AS. Dissociated gender-specific effects of recurrent seizures on GABA signaling in CA1 pyramidal neurons: role of GABA(A) receptors. J Neurosci. 2008;28:1557–1567. [PubMed]
  • Gardiner RM. The effects of hypoglycaemia on cerebral blood flow and metabolism in the newborn calf. J Physiol. 1980;298:37–51. [PubMed]
  • Gibbs J, Berkow G, Coulter D. GABAA receptor function in developing rat thalamic reticular neurons: Whole-cell recordings of GABA-mediated currents and modulation by benzodiazepines. J Neurophysiol. 1996;76:2568–2579. [PubMed]
  • Glass HC, Glidden D, Jeremy RJ, Barkovich AJ, Ferriero DM, Miller SP. Clinical neonatal seizures are independently associated with outcome in infants at risk for hypoxic-ischemic brain injury. J Pediatr. 2009;155:318–323. [PMC free article] [PubMed]
  • Glass HC, Poulin C, Shevell MI. Topiramate for the treatment of neonatal seizures. Pediatr Neurol. 2011;44:439–442. [PMC free article] [PubMed]
  • Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, Polin RA, Robertson CM, Thoresen M, Whitelaw A, Gunn AJ. on behalf of the CoolCap study group. Selective head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. Lancet. 2005;365:663–670. [PubMed]
  • Gomeza J, Ohno K, Hulsmann S, Armsen W, Eulenburg V, Richter DW, Laube B, Betz H. Deletion of the mouse glycine transporter 2 results in a hyperekplexia phenotype and postnatal lethality. Neuron. 2003;40:797–806. [PubMed]
  • Grigonis AM, Murphy EH. The effects of epileptic cortical activity on the development of callosal projections. Brain Res Dev Brain Res. 1994;77:251–5. [PubMed]
  • Harbert MJ, Tam EWY, Glass HC, Bonifacio SL, Haeusslein LA, Barkovich AJ, Jeremy RJ, Rogers EE, Glidden DV, Ferriero DM. Hypothermia is correlated with seizure absence in perinatal stroke. J Child Neurol. 2011;26:1126–1130. [PMC free article] [PubMed]
  • Hauser WA, Annegers JF, Kurland LT. Incidence of epilepsy and unprovoked seizures in Rochester, Minnesota: 1935–1984. Epilepsia. 1993;34:453–468. [PubMed]
  • Holmes G. Epilepsy in the Developing Brain: Lessons from the laboratory and clinic. Epilepsia. 1997;38:12–30. [PubMed]
  • Holopainen IE. Seizures in the developing brain: cellular and molecular mechanisms of neuronal damage, neurogenesis and cellular reorganization. Neurochem Int. 2008;52:935–47. [PubMed]
  • Isaeva E, Isaev D, Khazipov R, Holmes GL. Long-term suppression of GABAergic activity by neonatal seizures in rat somatosensory cortex. Epilepsy Res. 2009;87:286–9. [PMC free article] [PubMed]
  • Isaeva E, Isaev D, Savrasova A, Khazipov R, Holmes GL. Recurrent neonatal seizures result in long-term increases in neuronal network excitability in the rat neocortex. Eur J Neurosci. 2010;31:1446–1455. [PMC free article] [PubMed]
  • Jensen FE, Applegate CD, Holtzman D, Belin TR, Burchfiel JL. Epileptogenic effect of hypoxia in the immature rodent brain. Ann Neurol. 1991;29:629–637. [PubMed]
  • Jensen FE, Blume H, Alvarado S, Firkusny I, Geary C. NBQX blocks acute and late epileptogenic effects of perinatal hypoxia. Epilepsia. 1995;36:966–72. [PubMed]
  • Jensen FE. The role of glutamate receptor maturation in perinatal seizures and brain injury. Int J Dev Neurosci. 2002;20:339–47. [PubMed]
  • Kadam SD, Dudek FE. Neuropathogical features of a rat model for perinatal hypoxic-ischemic encephalopathy with associated epilepsy. J Comp Neurol. 2007;505:716–737. [PubMed]
  • Kalueff AV, Minasyan A, Keisala T, Kuuslahti M, Miettinen S, Tuohimaa P. Increased severity of chemically induced seizures in mice with partially deleted Vitamin D receptor gene. Neurosci Lett. 2006;394:69–73. [PubMed]
  • Kapur J, Macdonald RL. Postnatal development of hippocampal dentate granule cell gamma-aminobutyric acidA receptor pharmacological properties. Mol Pharmacol. 1999;55:444–452. [PubMed]
  • Kern SE. Challenges in conducting clinical trials in children: approaches for improving performance. Expert Rev Clin Pharmacol. 2009;2:609–617. [PMC free article] [PubMed]
  • Kim M, Yu ZX, Fredholm BB, Rivkees SA. Susceptibility of the developing brain to acute hypoglycemia involving A1 adenosine receptor activation. Am J Physiol Endocrinol Metab. 2005;289:E562–9. [PubMed]
  • Kleen JK, Sesqué A, Wu EX, Miller FA, Hernan AE, Holmes GL, Scott RC. Early-life seizures produce lasting alterations in the structure and function of the prefrontal cortex. Epilepsy Behav. 2011;22:214–9. [PMC free article] [PubMed]
  • Koh S, Jensen FE. Topiramate blocks perinatal hypoxia-induced seizures in rat pups. Ann Neurol. 2001;50:366–72. [PubMed]
  • Kojima-ishii K, Kure S, Ichinohe A, Shinka T, Narisawa A, Komatsuzaki S, Kanno J, Kamada F, Aoki Y, Yokoyama H, Oda M, Sugawara T, Mizoi K, Nakahara D, Matsubara Y. Model mice for mild-form glycine encephalopathy: behavioral and biochemical characterizations and efficacy of antagonists for the glycine binding site of N-methyl D-aspartate receptor. Pediatr Res. 2008;64:228–33. [PubMed]
  • Krall RL. State of the controlled clinical trial enterprise in the United States. Clin Pharmacol Ther. 2011;89:225–228. [PubMed]
  • Lanska MJ, Lanska DJ, Baumann RJ, Kryscio RJ. A population-based study of neonatal seizures in Fayette County, Kentucky. Neurology. 1995;45:724–732. [PubMed]
  • Laurén HB, Lopez-Picon FR, Korpi ER, Holopainen IE. Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus. J Neurochem. 2005;94:1384–94. [PubMed]
  • Lee CL, Hrachovy RA, Smith KL, Frost JD, Jr, Swann JW. Tetanus toxin-induced seizures in infant rats and their effects on hippocampal excitability in adulthood. Brain Res. 1995;677:97–109. [PubMed]
  • Lee CL, Hannay J, Hrachovy R, Rashid S, Antalffy B, Swann JW. Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy. Neuroscience. 107:71–84. [PubMed]
  • Legido A, Clancy RR, Berman PH. Neurologic outcome after electroencephalographically proven neonatal seizures. Pediatrics. 1991;88:583–596. [PubMed]
  • Leib SL, Kim YS, Black SM, Tureen JH, Tauber MG. Inducible nitric oxide synthase and the effect of aminoguanidine in experimental neonatal meningitis. J Infect Dis. 1998;177:692–700. [PubMed]
  • Leinekugel X, Medina I, Khaliliv I, Ben-Ari Y, Khazipov R. Ca2+ oscillations mediated by the synergistic excitatory actions of GABA(A) and NMDA receptors in the neonatal hippocampus. Neuron. 1997;18:243–255. [PubMed]
  • Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci U S A. 1997;94:9831–9835. [PubMed]
  • Liu H, Kaur J, Dashtipour K, Kinyamu R, Ribak CE, Friedman LK. Suppression of hippocampal neurogenesis is associated with developmental stage, number of perinatal seizure episodes, and glucocorticosteroid level. Exp Neurol. 2003;184:196–213. [PubMed]
  • Liu J, Lv F, Sun W, Tao C, Ding G, Karaplis A, Brown E, Goltzman D, Miao D. The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH. PLoS Genet. 2011;7:e1002294. [PMC free article] [PubMed]
  • Ljungberg MC, Sunnen CN, Lugo JN, Anderson AE, D’Arcangelo G. Rapamycin suppresses seizures and neuronal hypertrophy in a mouse model of cortical dysplasia. Dis Model Mech. 2009;2:389–98. [PubMed]
  • Lombroso CT. Neonatal seizures: gaps between the laboratory and the clinic. Epilepsia. 2007;48(S2):83–106. [PubMed]
  • LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron. 1995;15:1287–1298. [PubMed]
  • Malingre MM, Van Rooij LGM, Rademaker CMA, Toet MC, Ververs TFFT, van Kersteren C, de Vries LS. Development of an optimal lidocaine infusion strategy for neonatal seizures. Eur J Pediatr. 2006;165:598–604. [PubMed]
  • Marsh E, Brooks-Kayal AR, Porter B. Seizures and antiepileptic drugs: Does exposure alter normal brain development? Epilepsia. 2006;47:1999–2010. [PubMed]
  • Marsh E, Fulp C, Gomez E, Nasrallah I, Minarcik J, Sudi J, Christian SL, Mancini G, Labosky P, Dobyns W, Brooks-Kayal A, Golden JA. Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females. Brain. 2009;132:1563–1576. [PMC free article] [PubMed]
  • McBride MC, Laroia N, Guillet R. Electrographic seizures in neonates correlate with poor neurodevelopmental outcome. Neurology. 2000;55:506–513. [PubMed]
  • McCabe BK, Silveira DC, Cilio MR, Cha BH, Liu X, Sogawa Y, Holmes GL. Reduced neurogenesis after neonatal seizures. J Neurosci. 2001;21:2094–103. [PubMed]
  • McGowan JE, Haynes-Laing AG, Mishra OP, Delivoria-Papadopoulos M. The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets. Brain Res. 1995;670:283–8. [PubMed]
  • Meador KJ, Baker GA, Browning N, Clayton-Smith J, Combs-Cantrell DT, Cohen M, Kalayjian LA, Kanner A, Liporace JD, Pennell PB, Privitera M, Loring DW. NEAD Study Group. Cognitive function at 3 years of age after fetal exposure to antiepileptic drugs. N Engl J Med. 2009;16:1597–1605. [PMC free article] [PubMed]
  • Meikle L, Talos DM, Onda H, Pollizzi K, Rotenberg A, Sahin M, Jensen FE, Kwiatkowski DJ. A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J Neurosci. 2007;27:5546–5558. [PubMed]
  • Miller SP, Weiss J, Barnwell A, Ferriero DM, Latal-Hajnal B, Ferrer-Rogers A, Newton N, Partridge JC, Glidden DV, Vigneron DB, Barkovich AJ. Seizure-associated brain injury in term newborns with perinatal asphyxia. Neurology. 2002;58:542–548. [PubMed]
  • Monyer H, Seeburg PH, Wisden W. Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing. Neuron. 1991;6:799–810. [PubMed]
  • Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron. 1994;12:529–540. [PubMed]
  • Murray DM, Boylan GB, Ali I, Ryan CA, Murphy BP, Connolly S. Defining the gap between electrographic seizure burden, clinical expression and staff recognition of neonatal seizures. Arch Dis Child Fetal Neonatal Ed. 2008;93:F187–F191. [PubMed]
  • Narisawa S, Wennberg C, Millán JL. Abnormal vitamin B6 metabolism in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization. J Pathol. 2001;193:125–133. [PubMed]
  • Owens DF, Boyce LH, Davis MB, Kriegstein AR. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci. 16:6414–6423. [PubMed]
  • Painter MJ, Scher MS, Stein AD, Armatti S, Wang Z, Gardiner JC, Paneth N, Minnigh B, Alvin J. Phenobarbital compared with phenytoin for the treatment of neonatal seizures. N Engl J Med. 1999;341:485–489. [PubMed]
  • Pedras-Vasconcelos JA, Puig M, Sauder C, Wolbert C, Ovanesov M, Goucher D, Verthelyi D. Immunotherapy with CpG oligonucleotides and antibodies to TNF-alpha rescues neonatal mice from lethal arenavirus-induced meningoencephalitis. J Immunol. 2008;180:8231–8240. [PubMed]
  • Peters HC, Hu H, Pongs O, Storm JF, Isbrandt D. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat Neurosci. 2005;8:51–60. [PubMed]
  • Plotkin MD, Snyder EY, Hebert SC, Delpire E. Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA’s excitatory role in immature brain. J Neurobiol. 1997;33:781–95. [PubMed]
  • Polderman KH. Mechanisms of action, physiological effects, and complications of hypothermia. Crit Care Med. 2009;37:S186–S202. [PubMed]
  • Porter BE. Neurogenesis and epilepsy in the developing brain. Epilepsia. 2008;49 (S5):50–4. [PMC free article] [PubMed]
  • Price MG, Yoo JW, Burgess DL, Deng F, Hrachovy RA, Frost JD, Jr, Noebels JL. A triplet repeat expansion genetic mouse model of infantile spasms syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and adult cognitive and behavioral impairment. J Neurosci. 2009;29:8752–8763. [PMC free article] [PubMed]
  • Putnam TJ, Merritt HH. Experimental Determination of the Anticonvulsant Properties of Some Phenyl Derivatives. Science. 1937;85:525–526. [PubMed]
  • Rakhade SN, Jensen FE. Epileptogenesis in the immature brain: emerging mechanisms. Nat Rev Neurol. 2009;5:380–391. [PMC free article] [PubMed]
  • Rakhade SN, Klein PM, Huynh T, Hilario-Gomez C, Kosaras B, Rotenberg A, Jensen FE. Development of later life spontaneous seizures in a rodent model of hypoxia-induced neonatal seizures. Epilepsia. 2011;52:753–65. [PMC free article] [PubMed]
  • Raol YH, Lapides DA, Keating JG, Brooks-Kayal AR, Cooper EC. A KCNQ channel opener for experimental neonatal seizures and status epilepticus. Ann Neurol. 2009;65:326–336. [PMC free article] [PubMed]
  • Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008;22:659–661. [PubMed]
  • Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K. The K+/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature. 1999;397:251–255. [PubMed]
  • Rogawski M, Donevan SD. AMPA receptors in epilepsy and as target for antiepileptic drugs. In: Delgado-Escueta AV, Wilson WA, Olsen RW, Porter RJ, editors. Jasper’s basic mechanisms of the epilepsies, Advances in neurology. Lippincott, Williams & Wilkins; Philadelphia: 1999. pp. 947–963.
  • Romijn HJ, Hofman MA, Gramsbergen A. At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby? Early Hum Dev. 1991;26:61–7. [PubMed]
  • Ronen GM, Penney S, Andrews W. The epidemiology of clinical neonatal seizures in Newfoundland: A population-based study. J Pediatr. 1999;134:71–75. [PubMed]
  • Rossetti AO. What is the value of hypothermia in acute neurologic diseases and status epilepticus? Epilepsia. 2011;52:S64–S66. [PubMed]
  • Saliba RM, Annegers JF, Waller KD, Tyson JE, Mizrahi EM. Incidence of neonatal seizures in Harris county Texas, 1992–1994. Am J Epidemiol. 1999;150:763–769. [PubMed]
  • Santos NF, Marques RH, Correia L, Sinigaglia-Coimbra R, Calderazzo L, Sanabria ER, Cavalheiro EA. Multiple pilocarpine-induced status epilepticus in developing rats: a long-term behavioral and electrophysiological study. Epilepsia. 2000;41(S6):S57–63. [PubMed]
  • Sarkisian MR. Overview of the Current Animal Models for Human Seizure and Epileptic Disorders. Epilepsy Behav. 2001;2:201–216. [PubMed]
  • Sayin U, Sutula TP, Stafstrom CE. Seizures in the developing brain cause adverse long-term effects on spatial learning and anxiety. Epilepsia. 2004;45:1539–1548. [PubMed]
  • Scantlebury MH, Galanopoulou AS, Chudomelova L, Raffo E, Betancourth D, Moshe SL. A model of symptomatic infantile spasms syndrome. Neurobiol Dis. 2010;37:604–612. [PMC free article] [PubMed]
  • Scher MS, Hamid MY, Steppe DA, Beggarly ME, Painter MJ. Ictal and interictal electrographic seizure durations in preterm and term neonates. Epilepsia. 1993;34:284–288. [PubMed]
  • Schleiss MR. Nonprimate models of congenital cytomegalovirus (CMV) infection: gaining insight into pathogenesis and prevention of disease in newborns. ILAR J. 2006;47:65–72. [PubMed]
  • Shankaran S, Laptook AR, Ehrenkranz RA, McDonald SA, Donovan EF, Fanaroff AA, Poole WK, Wright LL, Higgins RD, Finer NN, Carlo WA, Duara S, Oh W, Cotton M, Stevenson DK, Stoll BJ, Lemons JA, Guillet R, Jobe AH. Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med. 2005;353:1574–1584. [PubMed]
  • Silva AV, Regondi MC, Cipelletti B, Frassoni C, Cavalheiro EA, Spreafico R. Neocortical and hippocampal changes after multiple pilocarpine-induced status epilepticus in rats. Epilepsia. 2005;46:636–42. [PubMed]
  • Singh NA, Westenskow P, Charlier C, Pappas C, Leslie J, Anderson VE, Saguinetti MC, Leppert MF. BFNC Physician Consortium. KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum. Brain. 2003;126:2726–2737. [PubMed]
  • Singh NA, Otto JF, Dahle EJ, Pappas C, Leslie JD, Vilaythong A, Noebels JL, White HS, Wilcox KS, Leppert MF. Mouse models of human KCNQ2 and KCNQ3 mutations for benign familial neonatal convulsions show seiures and neuronal plasticity without synaptic reorganization. J Physiol. 2008;586:3405–3423. [PubMed]
  • Sogawa Y, Monokoshi M, Silveira DC, Cha BH, Cilio MR, McCabe BK, Liu X, Hu Y, Holmes GL. Timing of cognitive deficits following neonatal seizures: relationship to histological changes in the hippocampus. Brain Res Dev Brain Res. 2001;131:73–83. [PubMed]
  • Stables JP, Bertram EH, White HS, Coulter DA, Dichter MA, Jacobs MP, Loscher W, Lowenstein DH, Moshe SL, Noebels JL, Davis M. Models for epilepsy and epileptogenesis: report from the NIH workshop, Bethesda, Maryland. Epilepsia. 2002;43:1410–1420. [PubMed]
  • Stafstrom CE, Thompson JL, Holmes GL. Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures. Dev Brain Res. 1992;65:227–236. [PubMed]
  • Stafstrom CE, Moshe SL, Swann JW, Nehlig A, Jacobs MP, Schwartzkroin PA. Models of pediatric epilepsies: strategies and opportunities. Epilepsia. 2006;47:1407–1414. [PubMed]
  • Stephenson T. How children’s responses to drugs differ from adults. Br J Clin Pharmacol. 2005;59:670–673. [PubMed]
  • Swann J, Brady R, Martin D. Postnatal development of GABA-mediated synaptic inhibition in rat hippocampus. Neuroscience. 1989;28:551–561. [PubMed]
  • Swann JW, Le JT, Lee CL. Recurrent seizures and the molecular maturation of hippocampal and neocortical glutamatergic synapses. Dev Neurosci. 2007;29:168–78. [PubMed]
  • Talos DM, Fishman RE, Park H, Folkerth RD, Follett PL, Volpe JJ, Jensen FE. Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex. J Comp Neurol. 2006;497:42–60. [PubMed]
  • Talos DM, Follett PL, Folkerth RD, Fishman RE, Trachtenberg FL, Volpe JJ, Jensen FE. Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. II. Human cerebral white matter and cortex. J Comp Neurol. 2006;497:61–77. [PMC free article] [PubMed]
  • Tao JD, Mathur AM. Using amplitude-integrated EEG in neonatal intensive care. J Perinatol. 2010;30:S73–S81. [PubMed]
  • Tekgul H, Gauvreau K, Soul J, Murphy L, Robertson R, Stewart J, Volpe J, Bourgeois B, du Plessis AJ. The current etiologic profile and neurodevelopmental outcome of seizures in term newborn infants. Pediatrics. 2006;117:1270–1280. [PubMed]
  • Toet MC, van der Meij W, de Vries LS, Uiterwaal CSPM, van Huffelen KC. Comparison between simultaneously recorded amplitude integrated electroencephalogram (cerebral function monitor) and standard electroencephalogram in neonates. Pediatrics. 2002;109:772–779. [PubMed]
  • Towfighi J, Housman C, Mauger D, Vannucci RC. Effect of seizures on cerebral hypoxic-ischemic lesions in immature rats. Dev Brain Res. 1999;113:83–95. [PubMed]
  • Tsutsui Y, Kosugi I, Kawasaki H. Neuropathogenesis in cytomegalovirus infection: indication of the mechanisms using mouse models. Rev Med Virol. 2005;15:327–45. [PubMed]
  • Tucker AM, Aquilina K, Chakkarapani E, Hobbs CE, Thoresen M. Development of amplitude-integrated electroencephalography and interburst interval in the rat. Pediatr Res. 2009;65:62–6. [PubMed]
  • Van Rooij LGM, de Vries LS, Handryastuti S, Hawani D, Groenendaal F, van Huffelen AC, Toet MC. Neurodevelopmental outcome in term infants with status epilepticus detected with amplitude-integrated electroencephalography. Pediatrics. 2007;120:e354–363. [PubMed]
  • Van Rooij LGM, Toet MC, van Huffelen AC, Groenendaal F, Laan W, Zecic A, de Haan T, van Straaten ILM, Vrancken S, van Wezel G, van der Sluijs J, ter Horst H, Gavilanes D, Laroche S, Naulaers G, de Vries LS. Effect of treatment of subclinical neonatal seizures detected with aEEG: randomized, controlled trial. Pediatrics. 2010;125:e358–366. [PubMed]
  • Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Südhof TC. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science. 2000;287:864–869. [PubMed]
  • Way SW, McKenna J, 3rd, Mietzsch U, Reith RM, Wu HC, Gambello MJ. Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse. Hum Mol Genet. 2009;18:1252–1265. [PMC free article] [PubMed]
  • Waymire KG, Mahuren JD, Jaje JM, Guilarte TR, Coburn SP, MacGregor GR. Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat Genet. 1995;11:45–51. [PubMed]
  • Watson RE, Desesso JM, Hurtt ME, Cappon GD. Postnatal growth and morphological development of the brain: a species comparison. Birth Defects Res B Dev Reprod Toxicol. 2006;77:471–84. [PubMed]
  • Wirrell EC, Armstrong EA, Osman LD, Yager JY. Prolonged seizures exacerbate perinatal hypoxic-ischemic brain damage. Pediatr Res. 2001;50:445–454. [PubMed]
  • Wusthoff CJ, Dlugos DJ, Gutierrez-Colina A, Wang A, Cook N, Donnelly M, Clancy R, Abend NS. Electrographic seizures during therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy. J Child Neurol. 2011;26:724–728. [PMC free article] [PubMed]
  • Xiu-Yu S, Ruo-Peng S, Ji-Wen W. Consequences of pilocarpine-induced recurrent seizures in neonatal rats. Brain Dev. 2007;29:157–63. [PubMed]
  • Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet. 1997;16:391–396. [PubMed]
  • Zhang G, Raol YH, Hsu FC, Coulter DA, Brooks-Kayal AR. Effects of status epilepticus on hippocampal GABAA receptors are age-dependent. Neuroscience. 2004a;125:299–303. [PMC free article] [PubMed]
  • Zhang G, Raol YH, Hsu FC, Brooks-Kayal AR. Long-term alterations in glutamate receptor and transporter expression following early-life seizures are associated with increased seizure susceptibility. J Neurochem. 2004b;88:91–101. [PubMed]
  • Zhou D, Qian J, Liu CX, Chang H, Sun RP. Repetitive and profound insulin-induced hypoglycemia results in brain damage in newborn rats: an approach to establish an animal model of brain injury induced by neonatal hypoglycemia. Eur J Pediatr. 2008;167:1169–1174. [PubMed]