Neonatal exposure to a neuroactive steroid alters low-frequency oscillations in the subiculum.

Preclinical studies have established that neonatal exposure to contemporary sedative/hypnotic drugs causes neurotoxicity in the developing rodent and primate brains. Our group recently reported that novel neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) induced effective hypnosis in both neonatal and adult rodents but did not cause significant neurotoxicity in vulnerable brain regions such as subiculum, an output region of hippocampal formation particularly sensitive to commonly used sedatives/hypnotics. Despite significant emphasis on patho-morphological changes, little is known about long-term effects on subicular neurophysiology after neonatal exposure to neuroactive steroids. Hence, we explored the lasting effects of neonatal exposure to 3ß-OH on sleep macrostructure as well as subicular neuronal oscillations in vivo and synaptic plasticity ex vivo in adolescent rats. At postnatal day 7, we exposed rat pups to either 10 mg/kg of 3ß-OH over a period of 12 h or to volume-matched cyclodextrin vehicle. At weaning age, a cohort of rats was implanted with a cortical electroencephalogram (EEG) and subicular depth electrodes. At postnatal day 30-33, we performed in vivo assessment of sleep macrostructure (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and power spectra in cortex and subiculum. In a second cohort of 3ß-OH exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) in adolescent rats. Overall, we found that neonatal exposure to 3ß-OH decreased subicular delta and sigma oscillations during non-rapid eye movement sleep without altering sleep macrostructure. Furthermore, we observed no significant changes in subicular synaptic plasticity. Interestingly, our previous study found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep and profoundly suppressed subicular LTP in adolescent rats. Together these results suggest that exposure to different sedative/hypnotic agents during a critical period of brain development may induce distinct functional changes in subiculum circuitry that may persist into adolescent age.

CaV3.1 T-type calcium channels are important for spatial memory processing in the dorsal subiculum.

The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering neuronal excitability by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide an important causative link between the CaV3.1 channels, burst firing of dSub neurons and memory formation, thus further supporting the notion that changes in neuronal excitability regulate memory processing. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.

Neonatal Isoflurane Does Not Affect Sleep Architecture and Minimally Alters Neuronal Beta Oscillations in Adolescent Rats.

General anesthetics are neurotoxic to the developing rodent and primate brains leading to neurocognitive and socio-affective impairment later in life. In addition, sleep patterns are important predictors of cognitive outcomes. Yet, little is known about how anesthetics affect sleep-wake behaviors and their corresponding oscillations. Here we examine how neonatal general anesthesia affects sleep and wake behavior and associated neuronal oscillations. We exposed male and female rat pups to either 6 h of continuous isoflurane or sham anesthesia (compressed air) at the peak of their brain development (postnatal day 7). One cohort of animals was used to examine neurotoxic insult 2 h post-anesthesia exposure. At weaning age, a second cohort of rats was implanted with cortical electroencephalogram electrodes and allowed to recover. During adolescence, we measured sleep architecture (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and electroencephalogram power spectra over a 24 h period. We found that exposure to neonatal isoflurane caused extensive neurotoxicity but did not disrupt sleep architecture in adolescent rats. However, these animals had a small but significant reduction in beta oscillations, specifically in the 12-20 Hz beta 1 range, associated with wake behavior. Furthermore, beta oscillations play a critical role in cortical development, cognitive processing, and homeostatic sleep drive. We speculate that dysregulation of beta oscillations may be implicated in cognitive and socio-affective outcomes associated with neonatal anesthesia.

Differential effects of the novel neurosteroid hypnotic (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile on electroencephalogram activity in male and female rats.

BACKGROUND: We recently showed that a neurosteroid analogue, (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH), induced hypnosis in rats. The aim of the present study was to evaluate the hypnotic and anaesthetic potential of 3ß-OH further using electroencephalography. METHODS: We used behavioural assessment and cortical electroencephalogram (EEG) spectral power analysis to examine hypnotic and anaesthetic effects of 3ß-OH (30 and 60 mg kg-1) administered intraperitoneally or intravenously to young adult male and female rats. RESULTS: We found dose-dependent sex differences in 3ß-OH-induced hypnosis and EEG changes. Both male and female rats responded similarly to i.p. 3ß-OH 30 mg kg-1. However, at the higher dose (60 mg kg-1, i.p.), female rats had two-fold longer duration of spontaneous immobility than male rats (203.4 [61.6] min vs 101.3 [32.1] min), and their EEG was suppressed in the low-frequency range (2-6 Hz), in contrast to male rats. Although a sex-dependent hypnotic effect was not confirmed after 30 mg kg-1 i.v., female rats appeared more sensitive to 3ß-OH with relatively small changes within delta (1-4 Hz) and alpha (8-13 Hz) bands. Finally, 3ß-OH had a rapid onset of action and potent hypnotic/anaesthetic effect after 60 mg kg-1 i.v. in rats of both sexes; however, all female rats and only half of the male rats reached burst suppression, an EEG pattern usually associated with profound inhibition of thalamocortical networks. CONCLUSIONS: Based on its behavioural effects and EEG signature, 3ß-OH is a potent hypnotic in rats, with female rats being more sensitive than male rats.

The T-type calcium channel isoform Cav3.1 is a target for the hypnotic effect of the anaesthetic neurosteroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile.

BACKGROUND: The mechanisms underlying the role of T-type calcium channels (T-channels) in thalamocortical excitability and oscillations in vivo during neurosteroid-induced hypnosis are largely unknown. METHODS: We used patch-clamp electrophysiological recordings from acute brain slices ex vivo, recordings of local field potentials (LFPs) from the central medial thalamic nucleus in vivo, and wild-type (WT) and Cav3.1 knock-out mice to investigate the molecular mechanisms of hypnosis induced by the neurosteroid analogue (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH). RESULTS: Patch-clamp recordings showed that 3ß-OH inhibited isolated T-currents but had no effect on phasic or tonic γ-aminobutyric acid A currents. Also in acute brain slices, 3ß-OH inhibited the spike firing mode more profoundly in WT than in Cav3.1 knockout mice. Furthermore, 3ß-OH significantly hyperpolarised neurones, reduced the amplitudes of low threshold spikes, and diminished rebound burst firing only in WT mice. We found that 80 mg kg-1 i.p. injections of 3ß-OH induced hypnosis in >60% of WT mice but failed to induce hypnosis in the majority of mutant mice. A subhypnotic dose of 3ß-OH (20 mg kg-1 i.p.) accelerated induction of hypnosis by isoflurane only in WT mice, but had similar effects on the maintenance of isoflurane-induced hypnosis in both WT and Cav3.1 knockout mice. In vivo recordings of LFPs showed that a hypnotic dose of 3ß-OH increased δ, θ, α, and ß oscillations in WT mice in comparison with Cav3.1 knock-out mice. CONCLUSIONS: The Cav3.1 T-channel isoform is critical for diminished thalamocortical excitability and oscillations that underlie neurosteroid-induced hypnosis.

The role of KCC2 in hyperexcitability of the neonatal brain.

BACKGROUND: The hyperpolarizing activity of γ-aminobutyric acid A (GABAA) receptors depends on the intracellular chloride gradient that is developmentally regulated by the activity of the chloride extruder potassium (K) chloride (Cl) cotransporter 2 (KCC2). In humans and rodents, KCC2 expression can be detected at birth. In rodents, KCC2 expression progressively increases and reaches adult-like levels by the second postnatal week of life. Several studies report changes in KCC2 expression levels in response to early-life injuries. However, the functional contribution of KCC2 in maintaining the excitation-inhibition balance in the neonatal brain is not clear. In the current study, we examined the effect of KCC2 antagonism on the neonatal brain activity under hyperexcitable conditions ex vivo and in vivo. METHODS: Ex vivo electrophysiology experiments were performed on hippocampal slices prepared from 7 to 9 days-old (P7-P9) male rats. Excitability of CA1 pyramidal neurons bathed in zero-Mg2+ buffer was measured using single-unit extracellular (loose) or cell-attach protocol before and after application of VU0463271, a specific antagonist of KCC2. To examine the functional role of KCC2 in vivo, the effect of VU0463271 on hypoxia-ischemia (HI)-induced ictal (seizures and brief runs of epileptiform discharges - BREDs), and inter-ictal spike and sharp-wave activity was measured in P7 male rats. A highly sensitive LC-MS/MS method was used to determine the distribution and the concentration of VU0463271 in the brain. RESULTS: Ex vivo blockade of KCC2 by VU0463271 significantly increased the frequency of zero-Mg2+-triggered spiking in CA1 pyramidal neurons. Similarly, in vivo administration of VU0463271 significantly increased the number of ictal events, BREDs duration, and spike and sharp-wave activity in HI rats. LC-MS/MS data revealed that following systemic administration, VU0463271 rapidly reached brain tissues and distributed well among different brain regions. CONCLUSION: The results suggest that KCC2 plays a critical functional role in maintaining the balance of excitation-inhibition in the neonatal brain, and thus it can be used as a therapeutic target to ameliorate injury associated with hyperexcitability in newborns.

Neonatal Ketamine Alters High-Frequency Oscillations and Synaptic Plasticity in the Subiculum But Does not Affect Sleep Macrostructure in Adolescent Rats.

Exposure to sedative/hypnotic and anesthetic drugs, such as ketamine, during the critical period of synaptogenesis, causes profound neurotoxicity in the developing rodent and primate brains and is associated with poor cognitive outcomes later in life. The subiculum is especially vulnerable to acute neurotoxicity after neonatal exposure to sedative/hypnotic and anesthetic drugs. The subiculum acts as a relay center between the hippocampal complex and various cortical and subcortical brain regions and is also an independent generator of gamma oscillations. Gamma oscillations are vital in neuronal synchronization and play a role in learning and memory during wake and sleep. However, there has been little research examining long-term changes in subicular neurophysiology after neonatal exposure to ketamine. Here we explore the lasting effects of neonatal ketamine exposure on sleep macrostructure as well as subicular neuronal oscillations and synaptic plasticity in rats. During the peak of rodent synaptogenesis at postnatal day 7, rat pups were exposed to either 40 mg/kg of ketamine over 12 h or to volume matched saline vehicle. At weaning age, a subset of rats were implanted with a cortical and subicular electroencephalogram electrode, and at postnatal day 31, we performed in vivo experiments that included sleep macrostructure (divided into the wake, non-rapid eye movement, and rapid eye movement sleep) and electroencephalogram power spectra in cortex and subiculum. In a second subset of ketamine exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) experiments in adolescent rats. Overall, we found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep but it did not alter sleep macrostructure. Also, we observed a significant decrease in subicular LTP. Gamma oscillations during non-rapid eye movement sleep are implicated in memory formation and consolidation, while LTP serves as a surrogate for learning and memory. Together these results suggest that lasting functional changes in subiculum circuitry may underlie neurocognitive impairments associated with neonatal exposure to anesthetic agents.

Effects of a potassium channel opener on brain injury and neurologic outcomes in an animal model of neonatal hypoxic-ischemic injury.

BACKGROUND: Hypoxia-ischemia (HI) is the most common cause of brain injury in newborns and the survivors often develop cognitive and sensorimotor disabilities that undermine the quality of life. In the current study, we examined the effectiveness of flupirtine, a potassium channel opener, shown previously in an animal model to have strong anti-neonatal-seizure efficacy, to provide neuroprotection and alleviate later-life disabilities caused by neonatal hypoxic-ischemic injury. METHODS: The rats were treated with a single dose of flupirtine for 4 days following HI induction in 7-day-old rats. The first dose of flupirtine was given after the induction of HI and during the reperfusion period. The effect of treatment was examined on acute and chronic brain injury, motor functions, and cognitive abilities. RESULTS: Flupirtine treatment significantly reduced HI-induced hippocampal and cortical tissue loss at acute time point. Furthermore, at chronic time point, flupirtine reduced contralateral hippocampal volume loss and partially reversed learning and memory impairments but failed to improve motor deficits. CONCLUSION: The flupirtine treatment regimen used in the current study significantly reduced brain injury at acute time point in an animal model of neonatal hypoxic-ischemic encephalopathy. However, these neuroprotective effects were not persistent and only modest improvement in functional outcomes were observed at chronic time points.

Novel neuroactive steroid with hypnotic and T-type calcium channel blocking properties exerts effective analgesia in a rodent model of post-surgical pain.

BACKGROUND AND PURPOSE: Neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) is a novel hypnotic and voltage-dependent blocker of T-type calcium channels. Here, we examine its potential analgesic effects and adjuvant anaesthetic properties using a post-surgical pain model in rodents. EXPERIMENTAL APPROACH: Analgesic properties of 3ß-OH were investigated in thermal and mechanical nociceptive tests in sham or surgically incised rats and mice, with drug injected either systemically (intraperitoneal) or locally via intrathecal or intraplantar routes. Hypnotic properties of 3ß-OH and its use as an adjuvant anaesthetic in combination with isoflurane were investigated using behavioural experiments and in vivo EEG recordings in adolescent rats. KEY RESULTS: A combination of 1% isoflurane with 3ß-OH (60 mg·kg-1 , i.p.) induced suppression of cortical EEG and stronger thermal and mechanical anti-hyperalgesia during 3 days post-surgery, when compared to isoflurane alone and isoflurane with morphine. 3ß-OH exerted prominent enantioselective thermal and mechanical antinociception in healthy rats and reduced T-channel-dependent excitability of primary sensory neurons. Intrathecal injection of 3ß-OH alleviated mechanical hyperalgesia, while repeated intraplantar application alleviated both thermal and mechanical hyperalgesia in the rats after incision. Using mouse genetics, we found that CaV 3.2 T-calcium channels are important for anti-hyperalgesic effect of 3ß-OH and are contributing to its hypnotic effect. CONCLUSION AND IMPLICATIONS: Our study identifies 3ß-OH as a novel analgesic for surgical procedures. 3ß-OH can be used to reduce T-channel-dependent excitability of peripheral sensory neurons as an adjuvant for induction and maintenance of general anaesthesia while improving analgesia and lowering the amount of volatile anaesthetic needed for surgery.

Methodologic recommendations and possible interpretations of video-EEG recordings in immature rodents used as experimental controls: A TASK1-WG2 report of the ILAE/AES Joint Translational Task Force.

The use of immature rodents to study physiologic aspects of cortical development requires high-quality recordings electroencephalography (EEG) with simultaneous video recording (vEEG) of behavior. Normative developmental vEEG data in control animals are fundamental for the study of abnormal background activity in animal models of seizures or other neurologic disorders. Electrical recordings from immature, freely behaving rodents can be particularly difficult because of the small size of immature rodents, their thin and soft skull, interference with the recording apparatus by the dam, and other technical challenges. In this report of the TASK1 Working Group 2 (WG2) of the International League Against Epilepsy/American Epilepsy Society (ILAE/AES) Joint Translational Task Force, we provide suggestions that aim to optimize future vEEG recordings from immature rodents, as well as their interpretation. We focus on recordings from immature rodents younger than 30 days old used as experimental controls, because the quality and correct interpretation of such recordings is important when interpreting the vEEG results of animals serving as models of neurologic disorders. We discuss the technical aspects of such recordings and compare tethered versus wireless approaches. We also summarize the appearance of common artifacts and various patterns of electrical activity seen in young rodents used as controls as a function of behavioral state, age, and (where known) sex and strain. The information herein will hopefully help improve the methodology of vEEG recordings from immature rodents and may lead to results and interpretations that are more consistent across studies from different laboratories.

Brain Distribution and Metabolism of Flupirtine, a Nonopioid Analgesic Drug with Antiseizure Effects, in Neonatal Rats.

Flupirtine, a nonopioid analgesic drug, is effective in treating neonatal seizures. However, its brain delivery and pharmacokinetics are unknown in neonatal mammals. The purpose of this study was to determine the pharmacokinetics of flupirtine and the formation of its active metabolite D-13223 in various tissues such as brain in neonate animals. On postnatal day 7, rat pups received 25 mg/kg of flupirtine intraperitoneally. Liver; heart; kidney; lung; spleen; retina; serum; and brain regions hippocampus, cortex, and the remaining brain (devoid of cerebellum) were harvested up to 24-h postdosing. An LC-MS/MS assay was developed to quantify flupirtine and D-13223. Flupirtine was delivered to all tissues assessed, with the highest area under the concentration vs. time curve (AUC0⁻24h) in liver (488 µg·h/g tissue) and the lowest in spleen (82 µg·h/g tissue). Flupirtine reached the brain, including the hippocampus and cortex, within 1 h of dosing and persisted at 24 h. Flupirtine AUC in various brain regions was approximately 195 µg·h/g tissue. The half-life of flupirtine in various tissues ranged from 3.1 to 5.2 h. D-13223 was formed in vivo and detected in all tissues assessed, with the concentrations being the highest in the liver. Incubation of isolated neonatal rat liver, heart, kidney, lung, spleen, whole eye, serum, or whole brain with flupirtine for 3 h at 37 °C formed D-13223 in all tissues, except serum. D-13223 formation was the highest in isolated liver tissue. Tissue partition coefficients based on isolated tissue uptake correlated well with in vivo tissue:serum drug exposure ratios. Thus, flupirtine reaches the target brain tissues from the systemic route in neonatal rats, and brain tissue forms the active metabolite D-13223.

Anticonvulsant effect of flupirtine in an animal model of neonatal hypoxic-ischemic encephalopathy.

Research studies suggest that neonatal seizures, which are most commonly associated with hypoxic-ischemic injury, may contribute to brain injury and adverse neurologic outcome. Unfortunately, neonatal seizures are often resistant to treatment with current anticonvulsants. In the present study, we evaluated the efficacy of flupirtine, administered at clinically relevant time-points, for the treatment of neonatal seizures in an animal model of hypoxic-ischemic injury that closely replicates features of the human syndrome. We also compared the efficacy of flupirtine to that of phenobarbital, the current first-line drug for neonatal seizures. Flupirtine is a KCNQ potassium channel opener. KCNQ channels play an important role in controlling brain excitability during early development. In this study, hypoxic-ischemic injury was induced in neonatal rats, and synchronized video-EEG records were acquired at various time-points during the experiment to identify seizures. The results revealed that flupirtine, administered either 5 min after the first electroclinical seizure, or following completion of 2 h of hypoxia, i.e., during the immediate reperfusion period, reduced the number of rats with electroclinical seizures, and also the frequency and total duration of electroclinical seizures. Further, daily dosing of flupirtine decreased the seizure burden over 3 days following HI-induction, and modified the natural evolution of acute seizures. Moreover, compared to a therapeutic dose of phenobarbital, which was modestly effective against electroclinical seizures, flupirtine showed greater efficacy. Our results indicate that flupirtine is an extremely effective treatment for neonatal seizures in rats and provide evidence for a trial of this medication in newborn humans.

EEG power as a biomarker to predict the outcome after cardiac arrest and cardiopulmonary resuscitation induced global ischemia.

AIMS: Cardiac arrest (CA) is a major cause of mortality and survivors often develop neurologic deficits. The objective of this study was to determine the effect of CA and cardiopulmonary resuscitation (CPR) in mice on the EEG and neurologic outcomes, and identify biomarkers that can prognosticate poor outcomes. MAIN METHODS: Video-EEG records were obtained at various periods following CA-CPR and examined manually to determine the presence of spikes and sharp-waves, and seizures. EEG power was calculated using a fast Fourier transform (FFT) algorithm. KEY FINDINGS: Fifty percent mice died within 72h following CA and successful CPR. Universal suppression of the background EEG was observed in all mice following CA-CPR, however, a more severe and sustained reduction in EEG power occurred in the mice that did not survive beyond 72h than those that survived until sacrificed. Spikes and sharp wave activity appeared in the cortex and hippocampus of all mice, but only one out of eight mice developed a purely electrographic seizure in the acute period after CA-CPR. Interestingly, none of the mice that died experienced any acute seizures. At 10days after the CA-CPR, 25% of the mice developed spontaneous convulsive and nonconvulsive seizures that remained restricted to the hippocampus. The frequency of nonconvulsive seizures was higher than that of convulsive seizures. SIGNIFICANCE: A strong association between changes in EEG power and mortality following CA-CPR were observed in our study. Therefore, we suggest that the EEG power can be used to prognosticate mortality following CA-CPR induced global ischemia.

Flupirtine effectively prevents development of acute neonatal seizures in an animal model of global hypoxia.

Current first-line drugs for the treatment of neonatal seizures have limited efficacy and are associated with side effects. Uncontrolled seizures may exacerbate brain injury and contribute to later-life neurological disability. Therefore, it is critical to develop a treatment for neonatal seizures that is effective and safe. In early-life, when the γ-aminobutyric acid (GABA) inhibitory system is not fully developed, potassium channels play an important role in controlling excitability. An earlier study demonstrated that flupirtine, a KCNQ potassium channel opener, is more efficacious than diazepam and phenobarbital for the treatment of chemoconvulsant-induced neonatal seizures. In newborns, seizures are most commonly associated with hypoxic-ischemic encephalopathy (HIE). Thus, in the present study, we examined the efficacy of flupirtine to treat neonatal seizures in an animal model of global hypoxia. Our results showed that flupirtine dose dependently blocks the occurrence of behavioral seizures in pups during hypoxia. Additionally, flupirtine inhibits the development of hypoxia-induced clinical seizures and associated epileptiform discharges, as well as purely electrographic (subclinical) seizures. These results suggest that flupirtine is an effective anti-seizure drug, and that further studies should be conducted to determine the time window within which it's administration can effectively treat neonatal seizures.

Effect of spontaneous seizures on GABAA receptor α4 subunit expression in an animal model of temporal lobe epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is frequently medically intractable and often progressive. Compromised inhibitory neurotransmission due to altered γ-aminobutyric acid (GABA)A receptor α4 subunit (GABAA Rα4) expression has been emphasized as a potential contributor to the initial development of epilepsy following a brain insult (primary epileptogenesis), but the regulation of GABAA Rα4 during chronic epilepsy, specifically, how expression is altered following spontaneous seizures, is less well understood. METHODS: Continuous video-electroencephalography (EEG) recordings from rats with pilocarpine-induced TLE were used to capture epileptic animals within 3 h of a spontaneous seizure (SS), or >24 h after the last SS, to determine whether recent occurrence of a seizure was associated with altered levels of GABAA Rα4 expression. We further evaluated whether this GABAA Rα4 plasticity is regulated by signaling mechanisms active in primary epileptogenesis, specifically, increases in brain-derived neurotrophic factor (BDNF) and early growth response factor 3 (Egr3). RESULTS: Elevated levels of GABAA Rα4 messenger RNA (mRNA) and protein were observed following spontaneous seizures, and were associated with higher levels of BDNF and Egr3 mRNA. SIGNIFICANCE: These data suggest that spontaneous, recurrent seizures that define chronic epilepsy may influence changes in GABAA Rα4 expression, and that signaling pathways known to regulate GABAA Rα4 expression after status epilepticus may also be activated after spontaneous seizures in chronically epileptic animals.

Repeated exposure to low doses of kainic acid activates nuclear factor kappa B (NF-κB) prior to seizure in transgenic NF-κB/EGFP reporter mice.

Predicting seizurogenic properties of pharmacologically active compounds is difficult due to the complex nature of the mechanisms involved and because of the low sensitivity and high variability associated with current behavioral-based methods. To identify early neuronal signaling events predictive of seizure, we exposed transgenic NF-κB/EGFP reporter mice to multiple low doses of kainic acid (KA), postulating that activation of the stress-responsive NF-κB pathway could be a sensitive indicator of seizurogenic potential. The sub-threshold dose level proximal to the induction of seizure was determined as 2.5mg/kg KA, using video EEG monitoring. Subsequent analysis of reporter expression demonstrated significant increases in NF-κB activation in the CA3 and CA1 regions of the hippocampus 24h after a single dose of 2.5mg/kg KA. This response was primarily observed in pyramidal neurons with little non-neuronal expression. Neuronal NF-κB/EGFP expression was observed in the absence of glial activation, indicating a lack of neurodegeneration-induced neuroinflammation. Protein expression of the immediate-early gene, Nurr1, increased in neurons in parallel to NF-κB activation, supporting that the sub-threshold doses of KA employed directly caused neuronal stress. Lastly, KA also stimulated NF-κB activation in organotypic hippocampal slice cultures established from NF-κB/EGFP reporter mice. Collectively, these data demonstrate the potential advantages of using genetically encoded stress pathway reporter models in the screening of seizurogenic properties of new pharamacologically active compounds.

Characterization of neonatal seizures in an animal model of hypoxic-ischemic encephalopathy.

OBJECTIVE: In this study, we use time-locked video and electroencephalography (EEG) recordings to characterize acute seizures and EEG abnormalities in an animal model that replicates many salient features of human neonatal hypoxic-ischemic encephalopathy (HIE) including the brain injury pattern and long-term neurologic outcome. METHODS: Hypoxia-ischemia (HI) was induced in 7-day-old rats by ligating the right carotid artery and exposing the pups to hypoxia for 2 h (Rice-Vannucci method). To identify seizures and abnormal EEG activity, pups were monitored by video-EEG during hypoxia and at various time points after HI. Occurrence of electroclinical seizures, purely electrographic seizures and other abnormal discharges on EEG, was quantified manually. A power spectrum analysis was done to evaluate the effects of HI on EEG spectra in the 1-50 Hz frequency band. RESULTS: During hypoxia, all pups exhibit short duration, but frequent electroclinical seizures. Almost all pups continue to have seizures in the immediate period following termination of hypoxia. In more than half of the HI rats, seizures persisted for 24 h; for some of them, the seizures continued for >48 h. Seizures were not observed in any rats at 72 h after HI induction. A significant reduction in background EEG voltage in the cortex ipsilateral to the ligated carotid artery occurred in rats subjected to HI. In addition, purely electrographic seizures, spikes, sharp waves, and brief runs of epileptiform discharges (BREDs) were also observed in these rats. SIGNIFICANCE: HI induction in P7 rats using the Rice-Vannucci method resulted in the development of seizures and EEG abnormalities similar to that seen in human neonates with HIE. Therefore, we conclude that this is a valid model to test the efficacy of novel interventions to treat neonatal seizures.

Plasma topiramate concentrations resulting from doses associated with neuroprotection against white matter injury and stroke in two strains of rat pups.

BACKGROUND: Cerebral white matter (WM) injury and stroke are common neuropathological injuries in newborns with congenital heart defects (CHDs) requiring surgery. Previous investigations in Long Evans rat pups subjected to hypoxia-ischemia found that intraperitoneal (i.p.) topiramate (TPM) at 30 mg/kg, but not 50 mg/kg, conferred neuroprotection. In Sprague-Dawley pups, a dose of 30 mg/kg protected against stroke. Concentrations associated with neuroprotective doses were not measured. The aims of this investigation were to determine concentrations associated with neuroprotective doses and to investigate the pharmacokinetics (PK) of i.p. TPM. METHODS: Concentration-time data following administration of 30 and 50 mg/kg doses were analyzed using nonlinear mixed-effect modeling. RESULTS: Mean predicted steady-state maximum and average concentrations following 30 mg/kg TPM were 31.3 and 16.8 µg/ml in Long Evans and 39.9 and 24.4 µg/ml in Sprague-Dawley pups. Mean predicted steady-state maximum and average concentrations following 50 mg/kg TPM were 52.1 and 28.1 µg/ml in Long Evans and 66.5 and 40.6 µg/ml in Sprague-Dawley pups. The apparent clearance (CL/F) and apparent volume of distribution (V/F) were 0.0470 ml/min and 22.2 ml, respectively, for Long Evans and 0.0325 ml/min and 19.7 ml, respectively, for Sprague-Dawley pups. CONCLUSION: TPM concentrations associated with neuroprotective doses were determined. Body size and strain were significant covariates on CL/F and V/F. Results provide targets for future neuroprotection studies.

Searching for new targets for treatment of pediatric epilepsy.

The highest incidence of seizures in humans occurs during the first year of life. The high susceptibility to seizures in neonates and infants is paralleled by animal studies showing a high propensity to seizures during early life. The immature brain is highly susceptible to seizures because of an imbalance of excitation and inhibition. While the primary outcome determinant of early-life seizures is etiology, there is evidence that seizures which are frequent or prolonged can result in long-term adverse consequences, and there is a consensus that recurrent early-life seizures should be treated. Unfortunately, seizures in many neonates and children remain refractory to therapy. There is therefore a pressing need for new seizure drugs as well as antiepileptic targets in children. In this review, we focus on mechanisms of early-life seizures, such as hypoxia-ischemia, and novel molecular targets, including the hyperpolarization-activated cyclic nucleotide-gated channels.

Neonatal seizures: controversies and challenges in translating new therapies from the lab to the isolette.

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 USA creating national collaborative project infrastructure, improved collaborative resources should advance clinical research on urgently needed new therapies for this disorder.