Research report
EGP-314 is expressed differentially in three brain zones at an early time in an experimentally induced ischemia rat model

https://doi.org/10.1016/j.molbrainres.2005.02.022Get rights and content

Abstract

Gene expression in frontal, occipital, and hippocampal regions of rat brains at 15 min of ischemic injury was studied in a rat model by producing focal cerebral ischemia through middle cerebral artery (MCA) occlusion without reperfusion. Catalase, epithelial glycoprotein (EGP-314), cytochrome C oxidase-subunit 1, ribosomal L31 protein, and ceruloplasmin were found to be differentially expressed. Specific primers were designed to study this newly reported brain EGP-314, a cellular adhesion molecule involved in cell–cell and cell–extracellular matrix interactions and related with cytoskeletal organization, differentiation, and proliferation. In the frontal and occipital lobes, EGP-314 expression was low in control and ischemic conditions and increased in sham injured conditions, whereas in the hippocampal region its expression was induced only by ischemia. In situ hybridization and immunohistochemistry revealed that EGP-314 mRNA and the protein were present in the ischemic hippocampus pyramidal neurons. DNA fragmentation was demonstrated by TUNEL and LM-PCR analysis in hippocampus region. TUNEL positive pyramidal neurons were observed at 15 min of ischemia. DNA ladder was found at 12 and 15 min of ischemia.

Introduction

Cerebral ischemia is caused by reduced blood supply to the brain tissue. Brain ischemia produces changes in oxygen metabolism, hormonal activity, nutrient concentrations, waste products disposal, and genetic expression, leading to cellular and molecular responses [32]. Altered gene expression is a prominent feature of ischemic cerebral injury and affects proteins of many functional classes. Expression of genes in the brain during ischemia has not been fully described but genes have been identified that express early after ischemia. These include genes from the fos and jun families and zif/268, which bind to specific sites of AP1 transcription factor [26], [36]. Actions of early genes include activation of the so-called late expression genes, which include effectors of protection and/or repair of damaged tissue. Among these late genes are neurotrophic factors, such as nervous growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 (NT-3, NT-4) [14], [18]. Besides, late expressed genes also include genes leading to cell destruction by apoptotic pathways [19].

EGP-314 is a monomeric glycoprotein of 314 amino acids located on the cell surface of human normal epithelial cells and it is considered a marker of the terminal plasma cells stage [6]. This molecule has been described repeatedly and has received different names (EGP, GA733-2, KSA, KS 1/4, MK1, CO17-IA, EP-CAM, ESA) due to its recognition by independently obtained monoclonal antibodies (mabs), although it has been proven that all of these molecules are the same [5]. The gene codifying for this molecule has been labeled as GA733-2 and there are orthologs described in humans and rats. In humans, EGP-314 expression has been observed in abnormally proliferating cells, such as in gastrointestinal adenocarcinoma, plasmocytoma, and breast cancer [9], it has been proposed to play a role in tumor metastasis [13], [35], it is associated with malignant proliferation, and in some cases its detection has been used as a marker for differential diagnosis [30]. This has led to test monoclonal antibodies directed against this molecule as part of experimental anti-tumor therapies [25].

EGP-314 has been proposed as a new adhesion type molecule, which is Ca2+ independent and negatively regulated by tumor necrosis factor α (TNFα) [14]. In addition, EGP-314 depicts homology with nidogen, laminin B1, and placental protein-12, which are matrix adhesion molecules [28]. The molecule is able to establish cell–cell or cell–matrix interactions and interacts with specific substrates through association with the actin cytoskeleton via α-actinin, suggesting a role in cell and tissue organization [2], [11], [22]. Interestingly, EGP-314 has two epidermal growth factor type II domains in the extracellular region that might function as a cell surface receptor capable of signal transduction [4].

Most cerebral ischemia studies have been performed in reperfusion models, in which damage was observed hours or days after ischemia induction [19], [35]. To contribute to the understanding of the immediate cerebral response to an ischemic insult, in this work surgically induced focal cerebral ischemia was produced in a rat model, which allowed us to study genes expression during the first 15 min of ischemia without reperfusion. The present work was aimed at assessing whether changes in EGP-314 expression occur in both mRNA and protein levels at an early time of focal cerebral ischemia.

Section snippets

Ischemic induction

Male Wistar rats weighing 250–450 g were anesthetized by inhalation of a mixture of oxygen and 2% halothane delivered by a Fluotec 3 machine (Ohmeda, West Yorkshire, England). Under a surgical microscope (Olympus Optical Co. LTD, Tokyo, Japan), focal cerebral ischemia of the left hemisphere was produced by introducing a 3-0 nylon monofilament 17 mm into the left internal carotid artery to occlude the middle cerebral artery (MCA) [37]. After 15 min of MCA occlusion, the rats were killed with a

Cerebral ischemia model

In this work, we analyzed the early expression of molecules, using focal cerebral ischemia induction by occlusion of the middle cerebral artery, which is a reliable model of reversible regional cerebral ischemia in rats without craniectomy [37]. Rat MCA is analogous to that in humans, it irrigates the parietal, frontal, and occipital lobes, and part of the hippocampus. In this model, it has been reported that permanent MCA occlusion produces neuronal injury in 37% of the coronal section area or

Acknowledgments

This project was supported by grants from CONACyT 756PM9506, MO-334. Alma Ortiz-Plata received a CONACyT scholarship (123714) during the course of the project. We thank Luis Miguel Salgado PhD and MSc. Ismael Vazquez Moctezuma for comments and technical advise and Biol. Leticia Cortes M for their technical assistance.

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