Elsevier

Epilepsy Research

Volume 68, Issue 2, February 2006, Pages 123-136
Epilepsy Research

Effect of neonatal isolation on outcome following neonatal seizures in rats—The role of corticosterone

https://doi.org/10.1016/j.eplepsyres.2005.10.005Get rights and content

Abstract

Emerging evidence indicates that early maternal care permanently modifies the activity of hypothalamic–pituitary–adrenal (HPA) axis and is a critical factor in determining the capacity of the brain to compensate for later encountered insults. The purpose of this study was to determine the role of corticosterone (CORT) in the detrimental effects of neonatal isolation (NI) on seizures. Rats were assigned randomly to the following five groups: (1) control (CONT) rats; (2) NI rats that underwent daily separation from their dams from postnatal day 2 (P2) to P9; (3) status epilepticus (SE) rats, induced by lithium-pilocarpine (Li-Pilo) model at P10; (4) NI plus SE (NIS) rats and (5) NISM rats, a subset of NIS rats receiving metyrapone (100 mg/kg), a CORT synthesis inhibitor, immediately after SE induction. At P10, plasma CORT levels were compared at baseline in CONT and NI rats and in response to Li-Pilo-induced SE among SE, NIS and NISM rats. We evaluated the spatial memory in the Morris water maze at P50∼55, the expression of hippocampal cyclic adenosine monophosphate (cAMP)-responsive element-binding protein phosphorylation at serine-133 (pCREBSer-133) at P55, hippocampal neuronal damage at P80 and seizure threshold at P100. The isolated rats exhibited higher CORT release in response to SE than non-isolated rats, and the NIS rats had greater cognitive deficits and decreased seizure threshold compared to the CONT, NI and SE groups. By contrast, the NISM group, compared to the NIS group, showed a normal CORT response to SE and better spatial memory but no difference in seizure threshold. Compared to the CONT group, the hippocampal pCREBSer-133 level was significantly reduced in all experimental groups (NI, SE, NIS, NISM) with no differences between groups. All rats were free of spontaneous seizures later in life and had no discernible neuronal loss in the hippocampus. Results in this model demonstrate repetitive NI enhances response of plasma CORT to SE, and exacerbates the neurological consequences of neonatal SE. Amelioration of neurological sequelae following reduction of the SE-induced excessive rise in plasma CORT implicates CORT in the pathogenesis of NI increasing the vulnerability to seizures.

Introduction

The immature brain is more susceptible to seizures yet less vulnerable to seizure-induced injury than the adult brain (Dube et al., 2001, Lai et al., 2002, Sarkisian et al., 1997, Schmid et al., 1999, Stafstrom et al., 1993). The patterns of neuronal injuries from seizures are age-specific; their extent and severity increase with age (Druga et al., 2005, Kubova et al., 2004, Sankar et al., 1998). Lithium-pilocarpine (Li-Pilo)-induced status epilepticus (SE) in adult rats produces the features of human temporal lobe epilepsy (TLE) in some aspects, such as hippocampal sclerosis. However, when SE is induced at postnatal day 10 (P10) – which is roughly equivalent to a human newborn (Romijn et al., 1991) – rats do not become epileptic (Dube et al., 2001, Priel et al., 1996) nor develop neuronal loss, interictal hypometabolism (Dube et al., 2000, Dube et al., 2001) or cognitive deficits (Zhang et al., 2004). While the immature brain appears to be less vulnerable to the adverse effects of prolonged seizures than the mature brain (Holmes et al., 2002), seizures early in life can be associated with later cognitive and behavioral disturbances, even in the absence of overt structural neuronal damage (Lynch et al., 2000, Majak and Pitkanen, 2004, Sayin et al., 2004, Stafstrom, 2002). In addition, Mathern et al. (1995) found that an initial precipitating injury, especially if occurring before age 5 years, was probably critical to the pathophysiological process of TLE. Given the increasing apparent age-specific brain damage from seizures, it is critical to determine under what circumstances early life seizures produce enduring neurological sequelae.

Considerable evidence demonstrates that the early environment modifies the development of hypothalamic–pituitary–adrenal (HPA) axis, subsequent brain function and behavior (Francis et al., 2002, Levine, 1994, Lissau and Sorensen, 1994, Liu et al., 1997, Matthews, 2002, McCormick et al., 1998, McEwen, 2000, Meaney, 2001, Plotsky and Meaney, 1993, Roceri et al., 2004, Weaver et al., 2004). Exposure of the developing brain to severe and/or prolonged stress may result in hyperactivity of the stress system with resultant hyperactivation of the HPA axis (upregulation of corticotrophin-releasing hormone (CRH) mRNA expression in the hypothalamus and amygdale) (Avishai-Eliner et al., 2002, Brunson et al., 2001, Hatalski et al., 1998, Plotsky and Meaney, 1993, Wadhwa et al., 2001), amygdala hyperfunction (fear reaction) (Meaney, 2001, Moriceau et al., 2004), and hippocampal dysfunction (defective glucocoticoid negative feedback, impaired learning and memory) (Liu et al., 1997; Matthews, 2002; McEwen, 2000). The importance of parental care as a mediator of the effects of early environmental adversity on neural development has been well established. In particular, normal maternal behavior is a key regulator during the stress hyporesponsive period (SHRP), lasting from neonatal day 2–3 until the second week of age, and influences the development of the HPA axis (Levine, 1994, Mathern et al., 1998, Sapolsky and Meaney, 1986, Weaver et al., 2004). Weaver et al. (2004) demonstrated that maternal behavior produces enduring alterations of DNA methylation at the glucocorticoid receptor (GR) gene promoter, and the offspring of mothers that exhibit less licking and grooming of pups, as adults, show less GR gene expression in the hippocampus, and an increase of plasma CORT response to acute stress. Furthermore, both early life stress and high CORT levels increase the risk for the development of seizures (Chadda and Devaud, 2004, Haut et al., 2003).

In most studies published to date, neonatal seizures have been induced in the experimental animals under normal handling and environmental conditions. For humans, most early life seizures occur in premature and sick neonates (Miller et al., 2002, Scher, 2003, Scher et al., 1993) who are hospitalized and separated from their mothers, usually in a stressful environment. We have previously showed that rats subjected to repetitive neonatal isolation (NI) can exacerbate cognitive deficits following recurrent seizures (Huang et al., 2002). The precise mechanism by which NI increases neuronal vulnerability to neonatal seizures is not clear. Although the stress response is a complex biochemical cascade involving the release of diverse chemicals that can affect various aspects of neuro-physiological processes, glucocorticoids (GCs) are thought to be the primary candidate for programming of the fetal HPA axis during early life experience (Matthews, 2002). In addition, excessive GCs increase neuronal vulnerability, particularly in the presence of concomitant excitatory challenges (Kaufer et al., 2004).

To address the importance of early life maternal–infant interaction on the development and vulnerability of the brain, we investigated the effects of NI on early life SE and explored the role of CORT on the pathogenesis of neurological sequelae associated with NI followed by seizures. In this study, we examined the influence of NI on plasma CORT levels at baseline and in response to seizures, and determined whether NI exacerbated neonatal seizure-induced long-term neurological damage, focusing on spatial learning and memory, and the hippocampal phosphorylated cyclic adenosine monophosphate (cAMP)-responsive element-binding protein at serine-133 (pCREBSer-133), an important transcription factor underlying learning and memory (Lonze and Ginty, 2002, Silva et al., 1998). Seizure thresholds, spontaneous seizures, and hippocampal neuronal loss were examined, too. We also evaluated whether the immediate suppression of the SE-induced rise in plasma CORT by metyrapone, a CORT synthesis inhibitor, can reverse the deleterious effects of NI on neonatal SE-induced long-term consequences.

Section snippets

Overview of experiments

To investigate whether NI compromises the developing brain following a prolonged seizure, and whether increased CORT after SE played a role in the deleterious effects of NI on SE, the rats were assigned randomly to five groups: control (CONT) rats, rats experiencing neonatal isolation (NI), rats subjected to Li-Pilo-induced SE (SE), rats subjected to NI plus SE (NIS), and a subset of NIS rats treated with metyrapone (NISM). We subjected rat pups to normal rearing (non-isolated) or NI (isolated)

Determine the test-dosage of metyrapone

In unpublished data, we found that that metyrapone given immediately after the induction of seizures exerted a pharmacological protection on cognitive deficits at both 100 mg/kg (NISM100) and 150 mg/kg (NISM150) with an optimal effect of 100 mg/kg, while metyrapone at 200 mg/kg (NISM200) had a mild detrimental effect. The time spent in the target quadrant (percentage) in the probe trial, a measure of strength of memory, was NIS = 28.02 ± 1.91%; NISM100 = 37.19 ± 2.70%; NISM150 = 35.2 ± 4.82%; NISM200 = 28.96 ± 

Discussion

The current study presents evidence that NI increases the acute response of plasma CORT to SE, and subjects the immature brain to subsequent seizure-induced injury. First, the study demonstrates that the experience of NI does not influence the plasma CORT levels 24 h after the last isolation paradigm, but enhances the acute CORT response to SE. Second, sole NI or SE early in life does not result in enduring behavioral alterations or hippocampal neuronal loss. However, repeated NI prolongs

Acknowledgements

This study was supported by the National Science Council, Taiwan (NSC-93-2320-B-182A-003), National Heath Research Institute, Taiwan (NHRI-EX91-8909BP), National Institutes of Health, USA (NS27984 and NS44295). Chi-Mei Hospital, Taiwan, grant number: CMFHR9452, and Chang-Gung Memorial Hospital, Taiwan, grant number: CMRPG83044.

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