Elsevier

Brain Research

Volume 1182, 28 November 2007, Pages 138-143
Brain Research

Research Report
Characterization of monocyte chemoattractant protein-1 expression following a kainate model of status epilepticus

https://doi.org/10.1016/j.brainres.2007.08.092Get rights and content

Abstract

Brain injury due to seizure induces a robust inflammatory response that involves multiple factors. Although the expression of chemokines has been identified as a part of this response, there are remaining questions about their relative contribution to seizure pathogenesis. To address this, we report the expression profile of the chemokine, monocyte chemoattractant protein-1 (MCP-1, CCL2), during kainate-induced seizure in the rat hippocampus. Furthermore, we compare MCP-1 expression to the temporal profile of blood–brain barrier (BBB) permeability and immune cell recruitment at the injury site, since both of these events have been linked to MCP-1. We find that BBB permeability increased prior to upregulation of MCP-1, while MCP-1 upregulation and immune cell recruitment occurred concurrently, 7–13 h after opening of the BBB. Our findings support the following conclusions: (1) BBB opening to large proteins does not require MCP-1 upregulation; (2) Leukocyte immigration is not sufficient to induce BBB opening to large proteins; (3) MCP-1 upregulation likely mediates recruitment of macrophages/microglia and granulocytes during seizure injury, thus warranting further investigation of this chemokine.

Introduction

Seizure activity triggers a significant inflammatory response that is integral to subsequent tissue injury. Key elements of this response have been identified for experimental and clinical cases of epilepsy (reviewed in Vezzani and Granata, 2005). Shortly after seizure onset, affected neurons and nearby microglia and astrocytes secrete pro-inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (De Simoni et al., 2000, Rizzi et al., 2003). Upregulation of these and other cytokines is followed by several downstream events, including increased blood–brain barrier (BBB) permeability to large proteins (Bolton and Perry, 1998, van Vliet et al., 2007), upregulation of adhesion molecules by neurovascular endothelial cells (Akiyama et al., 1994), expression of chemoattractant chemokines by neurons, astrocytes, and microglia (Kalehua et al., 2004, Lee et al., 2007), and recruitment of central nervous system (CNS) resident and peripheral immune cells at the injury site (Bolton and Perry, 1998, Dinkel et al., 2003).

Collectively, this process is of clear relevance to the pathogenesis of epilepsy. Treatments that non-specifically block the inflammatory response can exacerbate (Baik et al., 1999, Kunz and Oliw, 2001) or attenuate (Wallenstein, 1987, Akarsu et al., 1998) seizure severity, highlighting the notion that inflammation participates in tissue damage and protection. Further analysis of the factors outlined above has identified specific mechanisms by which inflammation contributes to tissue damage, even pointing to novel treatment strategies. As one example, secretion of IL-1β has been shown to increase synaptic glutamate and Ca2+ influx into affected neurons during seizure, while blockade of IL-1β signaling significantly reduces seizure severity (Viviani et al., 2003, Ravizza et al., 2006).

A second factor implicated in tissue damage is the chemokine, monocyte chemoattractant protein-1 (MCP-1, CCL2). Typically, chemokines such as MCP-1 facilitate homing of immune cells to damaged tissue in the periphery (Johnston and Butcher, 2002) and CNS (Bell et al., 1996, Babcock et al., 2003). In a recent study, Sheehan and colleagues demonstrate this relationship in the context of seizure, where mice lacking MCP-1 were shown to have reduced recruitment of microglia and monocytes to a seizure injury (Sheehan et al., 2007). Critically, mice deficient in MCP-1 also exhibited an overall reduction in neuron loss, and delivery of active MCP-1 was sufficient to restore immune cell recruitment and neuron death (Sheehan et al., 2007). While this study strongly supports an immune cell-dependent mechanism for tissue damage, additional studies suggest that MCP-1 itself can be neurotoxic when applied to either hippocampal cultures (Kalehua et al., 2004) or primary retinal mixed cultures (Nakazawa et al., 2007).

A third hypothesis not yet tested in the context of seizure is that MCP-1 contributes to tissue injury via modulation of the BBB. This possibility is highly relevant to seizure, as increased BBB permeability to albumins and other large proteins occurs in both clinical and experimental cases of epilepsy (van Vliet et al., 2007). Furthermore, a deliberate breach of the BBB by focal application of albumin (Seiffert et al., 2004), or systemic infusion of mannitol (van Vliet et al., 2007), has been shown to induce or exacerbate seizures, respectively. Outside the realm of seizure, there is evidence that MCP-1 acts as a modulator of BBB permeability. When added in high concentrations, MCP-1 increases BBB permeability to protein albumins in brain-endothelial/astrocyte cocultures and when infused intraparenchymally in vivo (Stamatovic et al., 2005). Additional in vitro studies suggest that MCP-1 alters expression of tight junction proteins in neurovascular endothelial cells at the BBB (Song and Patcher, 2004). However, in the context of seizure, it is unclear if MCP-1 regulates immune cell recruitment and BBB permeability, as no study to date has examined all three endpoints within the same model.

To rectify this, we compared the temporal profile of BBB permeability, MCP-1 protein expression, and immune cell recruitment following seizure activity in the rat hippocampus. We assessed these inflammatory endpoints following intraparenchymal infusion of the glutamatergic excitotoxin, kainic acid (KA), which induces status epilepticus seizures, preferentially damages Cornu Ammonis 3 (CA3) in the hippocampus (Nadler et al., 1980), and is an important model of temporal lobe epilepsy (Ben-Ari and Cossart, 2000).

Section snippets

Results

Microinfusion of KA into the hippocampus resulted in neuron death specific to regions CA2/3 (Fig. 1A), which was detectable in all animals assayed at 18 h post-KA (data not shown). Infusion of vehicle in the contralateral hemisphere induced no detectable CA3 cell loss at any of the time points assayed (Fig. 1B and data not shown). KA infusion induced an early, significant increase in BBB permeability to Evans blue (EB) dye at 1.5–4.5 h post-KA (Fig. 1, Fig. 2), which was detectable in all of

Discussion

This study describes the temporal profile of BBB permeability, MCP-1 protein expression, and immune cell recruitment following induction of status epilepticus seizures in the rat hippocampus. To date, each of these factors has been classified for different models of seizure, but no study has explored all three of these endpoints within a single seizure model. As such, it has not been possible to determine if these factors are related or occur as separate components of the inflammatory response.

Subjects

All procedures used in this study were approved by the Stanford University Administrative Panel on Laboratory Care and the Association for Assessment of Laboratory Animal Care and are in compliance with the National Institute Health Guide for the Care and Use of Laboratory Animals. Subjects were housed in standard conditions with a 12 h light–dark schedule. After 7 days of acclimation, animals were subject to stereotaxic surgery.

Stereotaxic surgery

Male Sprague–Dawley rats (250–300 g) were anesthetized with

Acknowledgments

This research was supported by the Adler Foundation and the Gunn Research Fund in Neuroscience. KSK was supported by DePauw University and the John and Janice Fisher Fund for Faculty Development.

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