Elsevier

Brain Research

Volume 788, Issues 1–2, 30 March 1998, Pages 223-231
Brain Research

Research report
A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions

https://doi.org/10.1016/S0006-8993(97)01552-7Get rights and content

Abstract

The systemic administration of kainate (10 mg/ml) into adult Wistar rats produces seizures and neurodegeneration. We have studied the effect of kainate administration on cPLA2 and COX-2 immunoreactivities after 3 days and 1, 2, 4 and 11 weeks. The cPLA2 immunoreactivity was increased in hippocampal neurons at 1 and 3 days after kainate injection suggesting that PLA2 may be involved in neurodegeneration. Increased cPLA2 and COX-2 immunoreactivities in astrocytes at 1, 2, 4 and 11 weeks after kainate injection indicate an adaptive astrocytic response that may be associated with gliosis.

Introduction

Kainic acid (KA), a cyclic analog of glutamate, is a potent convulsant and neurotoxin. When injected systemically or intracerebrally to rats, it evokes seizures and produces neuronal degeneration in limbic structures (especially the hippocampal CA3 and dentate hilus) 6, 26. The activation of KA receptors initiates Na+ influx whereas, KA receptors are relatively impermeable to Ca2+[1]. More recent studies report that KA increases neuronal Ca2+ uptake presumably through the activation of voltage-sensitive Ca2+ channels and/or the Na+, Ca2+ antiporter 2, 7. KA binding to KA receptors causes the release of glutamate from nerve terminals and KA blocks glutamate reuptake [23]. All these processes may lead to an enhanced concentration of extracellular glutamate, which by its excitotoxic action can produce neuronal injury 9, 28. The above studies on KA toxicity in brain suggest that the mechanism of neurotoxic action of KA is quite complex and requires detailed investigation.

Multiple forms of phospholipases A2 (PLA2) and cyclooxygenases (COX-1 and COX-2) are known to occur in brain tissue 8, 12, 29. cPLA2 is an 85 kDa cytosolic enzyme that catalyzes a receptor-mediated hydrolysis of arachidonic acid from membrane phospholipids 20, 12, 15. The conversion of arachidonic acid to prostaglandin (PG)H2 is catalyzed by COX enzymes; (PG)H2 is further metabolized to physiologically active prostaglandins (PG-D2, PG-E2 and PG-F2α), prostacyclin (PG-I2) and thromboxanes 1, 31, 32. PLA2 and COX-2 are target enzymes for non-steroidal anti-inflammatory drugs. These enzymes are normally expressed throughout the brain and are regulated by glutamate receptors and glucocorticoids 1, 27. Several recent studies have indicated that PLA2 and COX enzymes play an important role in the neurodegenerative processes associated with excitotoxic, ischemic, and traumatic brain injuries 24, 5, 11and neurodegenerative diseases 10, 11.

The purpose of the present investigation was to study the effect of intravenous injections of kainic acid on the immunohistochemical distribution of cPLA2 and COX-2 in the rat brain at various time intervals after neuronal injury.

Section snippets

Kainate injections

Twenty eight adult male Wistar rats weighing approximately 200 g were used for the present study of which 20 were injected with kainic acid. They were anesthetized with an intraperitoneal injection of 1.2 ml of 7% chloral hydrate, followed by the exposure of the left external jugular vein. Kainate (0.2 ml of a 10 mg/ml solution) was then injected into the vein over 4 min, and the skin wound closed. This resulted in seizures after the rats had recovered from the anesthesia. They were sacrificed

Distribution of cPLA2 and COX-2 in the normal hippocampus (Fig. 1A,B)

A light staining to cPLA2 (Fig. 1A) and COX-2 (Fig. 1B) was observed in the soma and apical dendrites of pyramidal neurons in CA1–3 and dentate granule neurons. The nuclei of these cells were unlabeled. Very few or no immunopositive glial cells were observed in the normal hippocampus.

Changes observed in Nissl sections after kainate injection

Changes in the hippocampus of the rat after intravenous kainate injection were described previously [21]. In the present experiments, we again confirmed that in Nissl-stained sections, some animals showed no

Discussion

The major finding of the present study is that cPLA2 immunoreactivity was markedly increased in neurons at 1 and 3 days after kainic acid injection, and that immunoreactivities of both cPLA2 and COX-2 were markedly increased in astrocytes at 1, 2, 4 and 11 weeks after injection.

The increased cPLA2 immunoreactivity in degenerating neurons 3 days after kainic acid injection suggests that this enzyme may be involved in neurodegeneration [11]. Since an increased expression of apolipoprotein D (apo

Acknowledgements

This work was supported by a grant from the National University of Singapore (RP960372). AAF and LAH are supported by NIH grants NS-10165 and NS-29441. We thank Professor David DeWitt from the Department of Biochemistry, Michigan State University for a generous supply of anti-COX-2 antibody, and Miss Leong Lo Ngah for excellent technical assistance. TLS is a research student at the National University of Singapore.

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