Temporal progression of evoked field potentials in neocortical slices after unilateral hypoxia–ischemia in perinatal rats: Correlation with cortical epileptogenesis

Highlights

• Early epileptogenesis following unilateral cystic-PHI is focal.

• Early focal hyperexcitability is masked by inhibition.

• Late epileptogenesis detected in contralateral non-cystic cortex.

• PHI without cystic lesions remains similar to controls.

Abstract

Infarcts of the neonatal cerebral cortex can lead to progressive epilepsy, which is characterized by time-dependent increases in seizure frequency after the infarct and by shifts in seizure-onset zones from focal to multi-focal. Using a rat model of unilateral perinatal hypoxia–ischemia (PHI), where long-term seizure monitoring had previously demonstrated progressive epilepsy, evoked field potentials (EFPs) were recorded in layers II/III of coronal neocortical slices to analyze the underlying time-dependent, network-level alterations ipsilateral vs. contralateral to the infarct. At 3 weeks after PHI, EFPs ipsilateral to the infarct were normal in artificial cerebrospinal fluid (ACSF); however, after blocking GABA_A receptors with bicuculline methiodide (BMI, 30 μM), the slices with an infarct were more hyperexcitable than slices without an infarct. At 3 weeks, contralateral PHI slices had responses indistinguishable from controls. Six months after PHI in normal ACSF, both ipsi- and contralateral slices from rats with cortical infarcts showed prolonged afterdischarges, which were only slightly augmented in BMI. These data suggest that the early changes after PHI are localized to the ipsilateral infarcted cortex and masked by GABA-mediated inhibition; however, after 6 months, progressive epileptogenesis results in generation of robust bilateral hyperexcitability. Because these afterdischarges were only slightly prolonged by BMI, a time-dependent reduction of GABAergic transmission is hypothesized to contribute to the pronounced hyperexcitability at 6 months. These changes in the EFPs coincide with the seizure semiology of the epilepsy and therefore offer an opportunity to study the mechanisms underlying this form of progressive pediatric epilepsy.

Introduction

Perinatal hypoxia–ischemia (PHI) is an important cause of pediatric epilepsy. After a latent period, epileptic seizures are thought to originate initially from regions near the infarct, but over time the seizures increase in frequency and begin to be generated at sites remote from the infarct (Morrell, 1991, Kotila and Waltimo, 1992, Reinecke et al., 1999, Morrell and deToledo-Morrell, 1999, Neville, 2010). Transcortical diaschisis of epileptic foci – or secondary epileptogenesis (Wilder, 2001) – is a common consequence of brain lesions, but the temporal evolution and underlying mechanisms are poorly understood (Von Monakow, 1969, Buchkremer-Ratzmann and Witte, 1997, Buchkremer-Ratzmann et al., 1998, Carrera and Tononi, 2014). Together, the increase in seizure frequency and development of new seizure-onset zones represent a core component of the progressive nature of acquired epilepsy, and thus require further understanding if we eventually hope to modify the epileptogenic disease process in children with acquired epilepsy.

Postnatal day 7 (P7) rat pups subjected to unilateral PHI (Rice et al., 1981) demonstrate many features of acquired pediatric epilepsy arising from perinatal stroke and other forms of PHI injury (Williams et al., 2004, Kadam and Dudek, 2007). This PHI model shows ipsilateral cortical infarcts with peri-infarct cortical dysplasia, microgyri and dysmorphic neurons (Kadam and Dudek, 2007), similar to some children with intractable epilepsy (Hadjipanayis et al., 1997, Jacobs et al., 2000, Gaggero et al., 2001). In a previous study, long-term, continuous, video-electroencephalogram (EEG) recording of seizures and interictal spikes with radiotelemetry showed that the initial epileptic seizures were unilateral and originated in the peri-infarct cortex (Kadam et al., 2010) at 2 months of age; however, at 6 months of age, the seizure onsets were often bilateral with an occasional contralateral onset. With EEG recordings, however, seizure onsets may appear bilateral when they are actually focal in nature, because fast transmission of the ictal wave across the corpus callosum can obscure the focal and unilateral nature of seizure initiation. In addition, it is possible that seizures are initiated at another site independent of those that were not recorded. The degree to which the contralateral side becomes independently epileptogenic in this model therefore remains unclear when considered in light of EEG data alone. The present studies, which included experiments on neocortical slices both ipsilateral and contralateral to the infarct, aimed to better understand the progressive electrophysiological changes underlying this previously reported evolution in electrographic seizure initiation, which had been ascertained with bilateral, continuous, long-term EEG recordings (Kadam et al., 2010).

The pharmacological blockade of GABA receptors in brain slice experiments essentially isolates the effects of changes in GABAergic inhibition from alterations in intrinsic cellular mechanisms and local excitatory circuits. In the present study, brain slices from the ipsilateral (i.e., cystic infarct) and contralateral (i.e., no cystic infarct) cortex of rats 3 weeks and 6 months after PHI (i.e., 1 and ⩾6-month-old rats) were studied in both normal artificial cerebrospinal fluid (ACSF) and after blockade of GABA_A-receptors with bicuculline methiodide (BMI), which allowed a preliminary assessment of the relative contribution of intrinsic neuronal properties and local excitatory circuits vs. alterations in GABA-mediated inhibition. The present experiments suggest that time-dependent alterations in both excitatory and inhibitory synaptic mechanisms contribute to the progression of epileptogenesis, both ipsilateral and contralateral to the infarct.