NIH Public Access
`Author Manuscript
`Epilepsia. Author manuscript; available in PMC 2015 July 01.
`Published in final edited form as:
`Epilepsia. 2014 July ; 55(7): 985–993. doi:10.1111/epi.12646.
`
`Characterization of neonatal seizures in an animal model of
`hypoxic-ischemic encephalopathy
`
`Dayalan Sampath1, Andrew M. White1, and Yogendra H. Raol1,*
`1Department of Pediatrics, Division of Neurology, School of Medicine, Translational Epilepsy
`Research Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
`
`SUMMARY
`
`Objective—In this study, we use time-locked video and electroencephalograph (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 hours (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 in the EEG were quantified manually. A power spectrum analysis was
`done to evaluate the effects of HI on EEG spectra in the 1 to 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 over half of the HI rats seizures persisted for 24 hours, for some of them, the seizures
`continued for more than 48 hours. Seizures were not observed in any rats at 72 hours 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 (BRED) 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.
`
`Keywords
`Epilepsy; EEG; video; electroclinical; hypoxia; ischemia
`
`*Address correspondence to: Yogendra Raol, 12850 E. Montview Blvd, Rm 3108, MS 8605, Aurora, CO 80045, Phone:
`303-724-4257, Yogendra.Raol@ucdenver.edu.
`DISCLOSURE OF CONFLICTS OF INTEREST
`None of the authors have any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in
`ethical publication and affirm that this report is consistent with those guidelines.
`
`ARGENTUM Exhibit 1150
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
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`INTRODUCTION
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`Seizures occur more often in the neonatal period than at any other time of human life. The
`most common cause of neonatal seizures is hypoxic-ischemic encephalopathy (HIE),1, 2 a
`serious condition with a suggested incidence of 1 to 8 cases per 1000 live births.3 Hypoxia-
`ischemia (HI) in neonates also results in injury to various brain regions4 and survivors of
`such injury can experience a multitude of neurological problems such as cerebral palsy,
`learning deficits and epilepsy.2, 5, 6 Clinical as well as basic science research studies suggest
`that seizures may exacerbate HI-induced brain injury and contribute to poor neurological
`outcome7–13 (but also see14, 15). Current antiepileptic drugs were developed using adult
`animal models and are not fully effective in treating neonatal seizures. Due to
`developmental differences, an immature brain may respond differently to an injury and a
`treatment than a mature brain. Therefore, to find the most effective treatment for a neonatal
`disease, it is imperative to test the efficacy of novel drugs in an animal model that not only
`accurately replicates the etiology and symptoms of a disease, but also the age of onset of the
`disease.
`
`The Rice-Vannucci model of HI-induction in the postnatal day 7 (P7) rat16 is widely used to
`study neonatal HIE. This model exhibits many of the salient features of human neonatal HIE
`such as the extent of brain injury,17–19 development of epilepsy in some, but not all animals
`in later life,20 and the learning and memory deficits.21 However, a complete characterization
`of acute seizures in this model is still lacking. In the current study, we use a synchronized
`video and electroencephalograph (EEG) recording technique to study characteristics of HI-
`induced neonatal seizures that occur during and shortly following the time of insult. For
`certain disease conditions such as epilepsy, continuous video-EEG recording has become the
`gold standard to confirm the presence of convulsive seizures and also to detect non-
`convulsive or subclinical seizures. Video-EEG is particularly useful in the assessment of
`neonates in whom a large percentage of seizures are subclinical and for whom
`differentiation of normal movements from ictal motor activity can be difficult if not
`impossible based on behavioral observation alone. Further, in neonates, there is often
`dissociation between clinical (behavioral) seizures and EEG phenomena (known as
`electroclinical uncoupling), resulting in resolution of behavioral manifestations of seizures
`despite on-going electrographic seizures. Because of this phenomenon, it has been observed
`that some drugs effectively stop behavioral seizures in neonates without stopping
`electrographic seizures. Therefore, the use of video-EEG is being increasingly employed in
`clinical settings to identify and manage neonatal seizures. However, because of technical
`challenges, EEG is rarely obtained in animal models of neonatal diseases. In the current
`study, we describe the characteristics of acute electroclinical seizures and the background
`EEG in the Rice-Vannucci P7 rat model of neonatal HIE. This information will be helpful in
`assessing how closely this model replicates the human neonatal HIE condition as well as the
`model’s validity for testing the efficacy of drugs used in the treatment of neonatal seizures.
`
`METHODS
`
`All animal procedures were performed according to the protocol approved by the
`Institutional Animal Care and Use Committee of the University of Colorado Anschutz
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`Medical Campus (UC-AMC). Also, all efforts were made to reduce animal suffering and the
`number of animals used. Timed pregnant Sprague-Dawley rats were obtained from Charles
`River Laboratories (Wilmington, MA). The pregnant rats were at the 14th day of gestation
`(E14) on arrival at the laboratory animal facility of the UC-AMC, and delivered the pups at
`E22 or E23. The litter size varied from 11 to 13 pups and the weights ranged between 12.5
`to 15 grams at postnatal day 6 (P6). Only male pups were used in the current study. The
`experiments were performed in a sequence depicted in figure 1.
`
`Electrode implantation
`
`To record electrical activity of the brain, P6 rats (n = 16) were implanted bilaterally in the
`parietal cortex with silver electrodes (0.008″ outer diameter; A-M Systems, Carlsborg, WA).
`The holes for implantation were made 2.5 mm behind the bregma and 3 mm lateral from
`midline sutures. The length of the electrodes from the bottom of the pedestal was 3 mm.
`This length assured that the recording electrode was sufficiently inside the brain, but not too
`deep (to decrease cortical damage), to obtain good quality signal (supplemental figure 1).
`Two electrode placement schemes were employed, differing only in the placement of the
`reference electrode. In the first, a common reference electrode was placed near the lambda
`over the left hemisphere; in the second, the active electrode in each hemisphere was
`referenced to a separate electrode positioned near the lambda in the same hemisphere as
`itself. Except for the seizure characterization data, which were obtained from both
`configurations and are presented in the table 1, all data were acquired using the second
`configuration. The electrode assembly was fixed to the skull with tissue adhesive and dental
`acrylic cement. The entire implantation procedure was performed under isoflurane
`anesthesia (2–4% for induction, initiated at 2%; 1–1.5% for maintenance). After the surgery,
`the rats were treated with an analgesic (0.1 mg/kg buprenorphine) once every 12 hours for
`48 hours.
`
`Hypoxia-ischemia induction
`HI was induced in P7 pups (n = 12) according to a published protocol.16,20 The pups were
`anesthetized with isoflurane and, after infusion of Marcaine (0.5%) at the incision site, a
`small longitudinal cut was made along the midline of ventral cervical skin. The right
`common carotid artery was then identified and double ligated with 4-0 polyglycolic acid
`suture. The incision was closed with 4-0 nylon Dermalon sutures or with skin glue. The
`entire operation lasted for 10 to 12 minutes. Following carotid ligation, the pups remained
`with the dam in a warm cage for 60 to 105 minutes. The pups were then separated and
`monitored for 30 minutes by video-EEG, following which they were exposed to hypoxia for
`2 hours in an airtight chamber that was filled with 8% oxygen and 92% nitrogen gas
`mixture. The oxygen content of the chamber was monitored using an oxygen sensor (Dräger
`Pac 7000, Pittsburg, PA) and was maintained between 8 and 8.3%. The temperature and
`humidity of the chamber were also tightly controlled and maintained at 36.5° Celsius and 60
`to 70% respectively as variations in these parameters can affect both mortality rate and
`extent of the HI-induced injury.
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`Video-EEG recording
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`EEG signals synchronized with digital video were recorded using the Stellate Harmonie
`system (Natus Medical, San Carlos, CA). The EEG data were collected with a sampling rate
`of 1000 Hz and stored on a hard disk for off-line analysis. P6 pups (n = 12) were implanted
`with electrodes and, at P7, underwent 30 minutes of baseline video-EEG recording prior to
`carotid ligation. Following ligation and prior to hypoxia, a second 30-minute video-EEG
`record was acquired. The pups were continuously monitored by video-EEG during hypoxia.
`The video-EEG recording was continued for 2 more hours after completion of hypoxia. To
`find out if the pups experienced acute spontaneous seizures following HI-induction, they
`were monitored by video-EEG for 4 hours a day (two 2 hour sessions separated by a 2 hour
`break, during which the pups were housed with the dam) 24, 48 and 72 hours after HI-
`induction. A separate group of rats whose carotid artery was not ligated and who were not
`exposed to hypoxia (control rats, n = 4) was monitored using video-EEG under the same
`conditions and for the same duration as HI rats.
`
`Seizure characterization
`
`Video-EEG records were analyzed by the first author of the current paper (DS) and all the
`events (electroclinical and purely electrographic seizures) identified by DS were checked for
`accuracy by a board certified clinical epileptologist (AW) and a researcher with experience
`in animal EEG analyses (YR). Electroclinical seizures were defined by an EEG pattern that
`differed from background in either amplitude, frequency or both, evolved over time and
`contained spikes or sharps lasting for 10 seconds or more and were associated with a change
`in the rat’s behavior. Electrographic seizures were defined as seizures observed in the EEG
`that were not associated with a behavioral correlate on video. Brief runs of epileptiform
`discharges (BRED) were defined as EEG patterns similar to an electrographic seizure with
`duration greater than 2 but less than 10 seconds. These might or might not have an
`associated change in the rat’s behavior.
`
`Power Spectrum Analysis
`
`The power spectra were determined using a Fast Fourier Transform (FFT) algorithm written
`using Visual Basic (Microsoft, Redmond, WA) subroutines. A rectangular window was used
`with epoch sizes of 8192 points (sampling rate = 1000 Hz) and frequency bins of 1 Hz
`spanning 1 to 50 Hz. To compare groups of epochs characterizing different animal states
`(e.g., baseline, ictal during hypoxia, interictal during hypoxia, interictal post-HI
`background), we identified at least 10 EEG epochs (contiguous or not) that were acquired
`during that state and were devoid of movement artifacts. The average of the power for the
`different epochs in each frequency bin was calculated for each of the two groups (animal
`states) to be compared. To investigate the change in the shape of the power distribution, the
`actual power was normalized. To statistically compare groups, the power within the
`frequency bins was integrated into delta (1–4 Hz), theta (4–8 Hz), alpha (8–13 Hz), beta
`(13–25 Hz), and gamma (25–50 Hz) bands. Comparisons were performed using methods
`described below.
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`Statistical Analysis
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`GraphPad Prism 5 statistical software (GraphPad Software Inc., San Diego, CA) was used
`for statistical analysis. Friedman test with Dunn’s test for multiple comparisons was used to
`compare integrated power spectral values of EEG obtained up to 72 hours after carotid
`ligation with the corresponding pre-ligation baseline value for each rat, and to analyze age-
`dependent changes in EEG power in the control rats. An unpaired t-test was used to identify
`statistical differences in integrated power spectral values of EEG at P10 between control and
`HI rats. A one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used to
`evaluate the effect of HI on spike or sharp wave activity. A p value of less than 0.05 was
`considered statistically significant.
`
`RESULTS
`
`Characteristics of hypoxia-ischemia induced seizures
`
`Seizures were not observed in the control rats during any video-EEG recording sessions. In
`the HI group of rats, seizures were not detected during baseline EEG recording or in the
`period between carotid ligation and exposure to hypoxia. However, all pups quickly
`developed behavioral seizures with a clear electrographic correlate (electroclinical seizure)
`upon exposure to the hypoxic environment following carotid artery ligation (n = 12, table 1).
`The behavioral seizures consisted of clonic seizures, tonic posturing of the trunk, tonic-
`clonic seizures, facial twitching and stiffening of the tail (video 1, supplemental material).
`The clonic and tonic seizures could involve all extremities or originate unilaterally. Those
`which did originate unilaterally did frequently generalize. The EEG activity associated with
`the electroclinical seizures showed an evolution of amplitude, frequency or both and
`contained spikes and sharps (figure 2A). The rats experienced frequent, typically short
`duration seizures during hypoxia (table 1). Two out of twelve pups (16%) also developed
`purely electrographic seizures during hypoxia at an average frequency of 1 seizure per hour
`(table 1).
`
`In the period immediately following hypoxia i.e., during the reperfusion period, 11 out of 12
`rats (91%) continued to have electroclinical seizures. These seizures, similar to seizures of
`the hypoxic period, were brief and frequent (table 1). The behavioral seizures, which
`correlated with a change in EEG activity, consisted of clonic, tonic and tonic-clonic seizures
`(figure 2B). Some of the rats (3/12) also developed purely electrographic seizures (table 1).
`Twenty-four hours after the initial insult, 66% of the rats (8/12) continued to exhibit
`electroclinical seizures (table 1). For these rats, both the seizure frequency and the total time
`seizing were lower than during the hypoxic and the reperfusion period (table 1). The
`behavioral seizures consisted of body jerks associated with tonic and clonic seizures (figure
`2C). Many of these rats (7/12) also manifested purely electrographic seizures (table 1). Only
`25% of HI rats (3/12) continued to have electroclinical seizures 48 hours following HI-
`induction (table 1). Neither electroclinical nor purely electrographic seizures were observed
`in any rats 72 hours after HI-induction (n = 12, table 1).
`
`To examine whether there was any change in the shape of the EEG power spectrum within
`the 1 to 50 Hz frequency band during the electroclinical seizures, the sum of the raw EEG
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`powers in each frequency bin was normalized to one. The normalized power was then
`compared between ictal and inter-ictal epochs of the hypoxia period. The results revealed
`that during the electroclinical seizures, the relative distribution of power shifts from slower
`to faster frequencies. Specifically, increases in power at about 9 and 25 Hz were observed
`during the ictal activity in both cortices (supplemental figure 2).
`
`Characteristics of background or inter-ictal EEG
`
`The baseline EEG of P7 rats consisted of low amplitude activity (~30 μV) and appeared
`continuous, with intermittent periods of voltage attenuation (no movement during this
`period) alternating with high voltage activities (no movement except occasional twitches).
`Some spike and sharp wave like activity was also observed in the baseline EEG record.
`These activities, which were not associated with any visible movement artifact, were high
`amplitude (3 times the average amplitude) sharply-contoured waveforms with duration 20–
`200 msec (supplemental figure 3A). However, because of the invasive nature of the
`recording technique used in this study, it is not possible to conclude with certainty whether
`these activities were physiologic or pathologic. A significant increase in the number of
`spikes and sharp waves occurred following HI-induction (supplemental figure 3 B, C). No
`such change was observed in control rats at the similar time in the recording session as for
`the HI rats. This suggests that the increased spike and sharp wave activity observed in HI
`rats resulted from HI-induced injury. Along with many individual spikes and sharp waves,
`BREDs (supplemental figure 4), which first appeared during the hypoxia period, were also
`observed in the inter-ictal EEG record. During hypoxia, the EEG of the majority of rats
`(10/12) contained BREDs, which continued to appear during the reperfusion period (table
`1). In some of the rats, unlike electroclinical and electrographic seizures, BREDs were
`observed even 72 hours after HI-induction. However, the frequency and the duration of
`BREDs continued to decline with increasing time from initiation of hypoxia (table 1).
`
`Severe voltage suppression of the background or inter-ictal EEG was evident in both
`cortices during hypoxia and in the period immediately following hypoxia, but it was more
`pronounced in the cortex ipsilateral to the ligated carotid artery (right cortex). Quantitative
`analyses showed a significant reduction in the integrated EEG power in the beta and gamma
`frequency bands during the inter-ictal period in the right cortex while undergoing hypoxia as
`compared to the power in the same frequency bands in the baseline EEG (figure 4). In the
`period immediately following hypoxia, a significant drop in power in the alpha and beta
`bands was observed in the right cortex (figure 4). The reduction in the power in the alpha
`band persisted for another 24 hours. Also, a significant decrease in the inter-ictal theta
`power was observed in the right cortex 24 hours after HI-induction (figure 4). By 72 hours
`after HI (at P10), the EEG power in all the frequency bands in the right cortex was similar to
`the power in the baseline EEG acquired at P7 (figure 4). However, the EEG power in 1 to 25
`Hz frequency band at 72 hours after HI was significantly lower than the EEG power in the
`control rats at P10 (figure 5). In the hemisphere contralateral to the ligated carotid artery
`(left cortex), power in the alpha, beta and gamma frequency bands in the inter-ictal period
`during hypoxia showed a decreasing trend, but was not significantly different from the
`baseline power (figure 4). Further, the EEG power at 72 hours after HI was higher than the
`power in the baseline EEG that was acquired at P7 (figure 4); it was, however, similar to the
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`EEG power of P10 control rats (figure 5). In fact, the developmental changes in the EEG
`power in the left cortex of HI rats were similar to that of the control rats (supplemental
`figure 5).
`
`Discussion
`
`To determine if an intervention, such as the administration of a drug, results in a significant
`change, it is best to, as accurately as possible, characterize the baseline (control) state so that
`deviations from that baseline can be identified. This paper is the first to describe the
`characteristics and evolution of both behavioral and EEG seizures over a 72 hour time frame
`in a rat model of neonatal HIE, derived using the Rice-Vannucci method. The uniqueness
`and accuracy of the descriptions were facilitated through the use of video monitoring that
`was time-locked with EEG. This technique allowed us to accurately correlate each EEG
`waveform to a rat’s behavior. Due to the size and thickness of the skull of a neonatal rat, and
`because they cannot be kept separate from the dam for a very long-time, it is technically
`very challenging to implant multiple electrodes and keep them intact for a prolonged period
`before significant deterioration of the EEG signal occurs. Even with these difficulties, EEG
`is still the most reliable method for identifying seizures, especially in neonates in whom it is
`difficult to differentiate uncoordinated movement from behavioral seizure activity. Further,
`EEG is necessary to detect subclinical seizures, which are common in neonates with HIE.
`Thus, using the synchronized video-EEG technique, we demonstrated that induction of HI in
`P7 rats by the Rice-Vannucci method16, a commonly used animal model to study neonatal
`HIE, resulted in the development of recurrent short duration electroclinical seizures, purely
`electrographic seizures as well as abnormalities in background EEG.
`
`To be an accurate model of the human condition, we need to demonstrate that seizures seen
`in the Rice-Vannucci model are similar to those experienced by human neonates. In human
`neonates, seizures are often brief and repetitive22 and consist of clonic, tonic, myoclonic and
`subtle seizure subtypes.23–25 In our study, clonic, tonic and tonic-clonic seizures that were
`clearly associated with changes in the EEG activity were observed following the
`introduction of pups to the hypoxic environment. Myoclonic jerks, often preceded by a spike
`in the EEG, were also observed along with other seizure activity and, most commonly
`occurred just before an electroclinical seizure began. In this paper, we significantly expand
`on the current literature. In a study similar to our own, Hayakawa and colleagues26 also
`observed generalized tonic and tonic-clonic seizures associated with bursts of polyspikes or
`spike-and-wave complexes during hypoxia in P6 rats whose left carotid artery was ligated
`(Rice-Vannucci method). A recent study documented that induction of HI in P12 rats (P11,
`if the day of birth is considered as P0) by Rice-Vannucci method results in the development
`of behavioral seizure activity such as forelimb or hindlimb paddling which correlated with
`alterations in EEG activity during hypoxia.27 However, these studies did not determine
`whether seizures persisted following HI-induction. Electroclinical seizures consisting of
`myoclonic jerks, tonic posturing, clonic convulsions, cycling movements and generalized
`tonic-clonic episode have been observed in P7 rats,28,29 P10 rats,28 P12 mice,30 newborn
`piglets,12 and fetal sheep31 in which hypoxia/ischemia was induced by methods that were
`different from the Rice-Vannucci method.
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`Our study demonstrates that following HI-induction, electroclinical seizures occurred
`spontaneously for 24 hours in the majority and for 48 hours in 25% of HI rat pups. In human
`patients with neonatal HIE, first seizures (sub-clinical or electroclinical) have been observed
`6 to 20 hours after birth, and these seizures often continue to occur for 48 hours or
`more.32,33 It is important to note that because of practical reasons, human EEG monitoring
`usually does not begin before many hours have passed after the birth. Similar to
`electroclinical seizures, in our study, electrographic seizures were first observed during
`hypoxia and continued to appear for 24 hours after HI-induction. However, unlike
`electroclinical seizures, which occurred in almost all of the HI rats, electrographic seizures
`were observed in only a few of the HI rats. In human neonates subclinical or electrographic
`seizures without clinical correlates are common, especially subsequent to antiepileptic
`treatment.34
`
`Along with electroclinical and electrographic seizures, multiple abnormalities in the EEG
`background activities were observed in the HI animals. The changes observed in the EEG
`background activities have been used as a diagnostic tool to prognosticate long-term
`outcome.35 The baseline EEG of P7 rats and the EEG of control rats in our study appeared
`continuous, contained spikes and sharp wavelike discharges as well as alternate periods of
`suppression and higher amplitudes. Following HI-induction, a visually obvious increase in
`the number of spikes and sharp waves was observed. In a study of human neonates by
`Clancy and Legido, the presence of spikes or sharp waves at 3-month follow-up was
`associated with a 100% rate of postnatal epilepsy development, whereas only 27% patients
`without spikes or sharp waves developed postnatal epilepsy.36 Another abnormality in the
`EEG background that is commonly observed in the neonates with HIE and has a very strong
`prognostic value is voltage attenuation. In our study there was a significant and persistent
`attenuation of inter-ictal EEG voltage following the introduction of the pups to hypoxia after
`carotid ligation. In the cortex ipsilateral to ligated carotid artery of the HI rats, the EEG
`power at P10 was significantly lower than that of P10 control rats, which may suggest a
`delay in EEG maturation (dysmaturity). In both rats and humans, a severely depressed EEG
`background that persists for a period of time is considered to be indicative of severe brain
`damage and a marker for an abnormal long-term neurologic outcome. According to a study
`carried out by Murray and colleagues, an EEG with background suppression (> 50%
`reduction from the normal values) in neonates with HIE is associated with abnormal
`outcomes.37 Their study also suggests that if the amplitude remains attenuated at 48 hours
`after the birth, the prognosis is very poor.
`
`There are some limitations to the current study. Due to the age of the animals, they cannot
`be kept separate from the dam for a very long duration and therefore, prolonged continuous
`video-EEG record could not be acquired. As a result, we could have underestimated the
`frequency of recurring seizures, the percentage of animals that develop acute spontaneous
`seizures following HI-induction, and the length of the period during which seizures acutely
`recurred after the initial injury. In our study, because of the size of the skull and the animal,
`we could efficiently record from only two brain regions (more electrodes will increase the
`size and the weight of the implant). Hence, we could have missed those seizures that
`originated from deep brain structures and remained focal. In future studies, continuous
`monitoring may be achieved through the use of a wireless telemetry system to record EEG.
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`In a recent publication, Zayachkivsky and colleagues describe the features of a novel
`wireless telemetry system that was well-tolerated by the pups and the EEG was recorded
`continuously from the pups while they were housed with the dam.29 While this system
`allows for more continuous monitoring, it is however limited in that there is only one active
`recording electrode and therefore, EEG signals from multiple areas cannot be obtained.
`
`In this paper, not only have we shown that induction of HI in P7 rats by the Rice-Vannucci
`method replicates most of the features of acute seizures and EEG abnormalities associated
`with the human condition, but we have also, through direct EEG, power spectra, and video
`observation and analysis, sufficiently characterized the baseline model results such that we
`can compare these with a perturbed system to determine the impact of drugs or other
`interventions. Since this model also replicates chronic changes observed in human HIE
`condition, it can be used to study the effects of the treatment of acute seizures on chronic
`brain injury and long-term neurologic outcome. A shorter latency to acute seizures has been
`shown to directly correlate with the extent of the acute lesion in the Rice-Vannucci model of
`neonatal HIE.27 Similarly, long-term studies may help explain the relationship between
`acute seizures and the chronic effects of neonatal HIE.
`
`Supplementary Material
`
`Refer to Web version on PubMed Central for supplementary material.
`
`Acknowledgments
`
`This work is supported by NIH/NICHD R01 HD065534 grant (YHR), CURE Prevention of Acquired Epilepsy
`award (YHR) and NIH/NINDS K08 NS053610-05 grant (AMW). We thank the University of Colorado Anschutz
`Medical Campus Rodent In Vivo Neurophysiology Core for providing facilities to acquire and review video-EEG
`data. We also thank Dr. Michael Hall and the Neuroscience Core Machine Shop for help with the construction of
`hypoxia chamber. We thank Dr. Zhaoxing Pan, Research Institute, Children’s Hospital Colorado, for help with
`statistical analyses, and Dr. Marco Gonzalez and Philip Lam for their help with histology. The authors also wish to
`thank staff members of the University of Colorado Anschutz Medical Campus Biorepository Core Facility for their
`assistance with brain imaging (the facility is supported by NIH/NCATS Colorado CTSI Grant Number UL1
`TR001082).
`
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`Epilepsia. Author manuscript; available in PMC 2015 July 01.
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`NIH-PA Author Manuscript
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