Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death

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Abstract

Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.

Introduction

Excitotoxicity is involved in acute and chronic neurodegenerative conditions such as hypoxia-ischemia, status epilepticus and amyotrophic lateral sclerosis [1]. In experimental animals, excitotoxicity has been known to induce acute neuronal death (AND) and delayed neuronal death (DND). AND is caused by glutamate receptor-mediated mechanisms within a few days following excitotoxic insult [2], [3]. DND occurs several days after excitotoxic insults [4], [5]. Both AND and DND are induced by apoptotic and/or necrotic mechanisms [6], [7].

Prostanoids, such as prostaglandins (PGs) and thromboxanes, are arachidonic acid-derived lipid mediators that play important roles in neurodegenerative conditions [8], [9], [10]. However, there have been conflicting studies on the effect of non-steroidal anti-inflammatory drug (NSAID) treatment on KA-induced excitotoxicity. Some investigators reported anti-neurotoxic effects of non-steroidal anti-inflammatory drugs (NSAIDs) [11], [12], [13], [14], while others reported neurotoxic effects [15]. Furthermore, other studies found no effect of NSAIDs on hippocampal cell loss [14], [16].

In our previous studies, we invented a comprehensive quantification method for the simultaneous determination of accurate tissue contents of a wide variety of prostanoids using liquid chromatography-electrospray ionization–tandem mass spectrometry (LC-ESI–MS/MS) [17], [18]. We demonstrated that PGs are produced in the hippocampus with a biphasic time course following a single systemic treatment of KA in 3-week-old juvenile rats [19]. PG production during the initial phase, which is characterized by a sharp surge in the PG level occurring approximately 30 min after KA treatment, is relatively specific to the hippocampus, dependent on the KA receptor-mediated pathway, and catalyzed by constitutively expressed cyclooxygenase (COX)-1 and COX-2. PG production during the late phase, which is characterized by a sustained moderate elevation in PG level between 3 h and 24 h after KA treatment, is primarily catalyzed by induction of COX-2.

In the present study, we found that a single KA treatment produced chronic progressive neuronal death in the hippocampus of 3-week-old juvenile rats, which has not been previously documented in adult rats [20], [21]. These results may indicate a degree of vulnerability to excitotoxicity in hippocampal neurons in rats of this particular age. To investigate the mechanism of KA-induced neuronal death, we performed immunohistochemistry (IHC) using antibodies for cathepsin D and spectrin breakdown product (SBDP) 150, as indicators of necrosis, as well as apoptosis-inducing factor (AIF), an indicator of apoptosis. To evaluate the activation and proliferation status of glial cells, we assessed microglia and astrocytes by IHC for ionized calcium binding adaptor molecule 1 (Iba-1) and glutamine synthetase, respectively. We aimed to determine the biological significance of biphasic PG production in the hippocampus following excitotoxic insult with a special emphasis on the differentiation between acute and chronic neuronal death.

Section snippets

Animals

Three-week-old male Wistar rats (SLC, Hamamatsu, Japan), weighing between 33 and 37 g (mean, 35 g), were used. All animals were handled in accordance to the Guide for Animal Experiments at our institute, as well as the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, USA). Rats were housed at a temperature of 23±2 °C and 50±10% humidity, with a fixed 12-h light–dark cycle and free access to tap water and regular diet (CE-2, Clea, Tokyo, Japan). KA and

Time-dependent changes in the tissue contents of prostanoids

The tissue contents of a wide variety of prostanoids were quantified using an LC-ESI–MS/MS system at various time points following KA treatment, and the results of representative prostanoids are shown in Fig. 1. The hippocampal prostanoid levels rapidly peaked 30 min after KA treatment and then rapidly decreased (“initial phase” of PG production). The levels remained above basal levels for up to 24 h (“late phase”). The biphasic pattern of PG production (initial and late phases) in response to KA

Discussion

Here, we demonstrated that a single systemic treatment of KA induced progressive cell death of hippocampal pyramidal neurons over a 30-day period in juvenile rats. In addition to the well-described AND and DND, neuronal death continued in the CA1 even on day 30 after a single KA treatment, when the relative number of dying neurons was at its greatest in this study. Chronic progressive neuronal death, such as this, has not been well studied. Most investigators have not gone beyond 10 days when

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Contract grant numbers: 19790766 to K.Y., and Specially Promoted Research to T.S.) and as a part of the Rational Evolutionary Design of Advanced Biomolecules (REDS3) Project, Central Saitama Area, in the Program for Fostering Regional Innovation (City Area Type) from MEXT. The authors are thankful to Dr. Tamada (Senju Pharmaceuticals, Kobe, Japan) for providing anti-SBDP

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