Expression Analysis Systematic Explorer (EASE) analysis reveals differential gene expression in permanent and transient focal stroke rat models

Abstract

To gain greater insight on the molecular mechanisms that underlie ischemic stroke, we compared gene expression profiles in transient (tMCAO) and permanent middle cerebral artery occlusion (pMCAO) stroke models using Expression Analysis Systematic Explorer (EASE) pathway analysis software. Many transcripts were induced in both stroke models, including genes associated with transcriptional pathways, cell death, stress responses and metabolism. However, EASE analysis of the regulated genes indicated molecular functions and biological processes unique to each model. Pathways associated with tMCAO included inflammation, apoptosis and cell cycle, while pMCAO was associated with the induction of genes encoding neurotransmitter receptors, ion channels, growth factors and signaling molecules. An intriguing finding was the involvement of tyrosine kinases and phosphatases following pMCAO. These results provide evidence that neuronal death following tMCAO and pMCAO involves distinct mechanisms. These findings may give new insight to the molecular mechanisms involved in stroke and may lead to novel neuroprotective strategies.

Introduction

Ischemic stroke is a major cause of death and invalidity in Western society. However, mechanisms involved in ischemia-induced brain injury remain poorly understood. The development of strategies to treat acute stroke has been unsuccessful in clinical trials, therefore, new technologies for investigating the mechanisms involved in stroke may be useful to elucidate novel approaches to design beneficial therapeutic treatments for stroke. Much of the current knowledge regarding the development, function and pathophysiology of the central nervous system (CNS) has evolved from the traditional “one function, one gene” approach that investigates the manipulation of a single gene or few genes in order to study neuropathology and to discover potential therapeutic strategies. However, it is clear that complex CNS cellular functions involve the simultaneous and concerted induction and repression of multiple genes. With the completion of the sequence of the human genome and the increasing number of genomic sequences available for other model organisms, it is now possible to simultaneously examine the expression of thousands of transcripts in cells and tissues (Lipshutz et al., 1999, Schulze and Downward, 2001).

Several previous studies have examined gene expression profiles in brain tissues following ischemia in rodent stroke models using microarray technology or similar means to elucidate the mechanisms involved in the neuronal death that follows ischemia (Bates et al., 2001, Bowler et al., 2002, Lu et al., 2003, Lu et al., 2004, Raghavendra Rao et al., 2002, Schmidt-Kastner et al., 2002, Schroeter et al., 2003, Soriano et al., 2000, Stenzel-Poore et al., 2003). These studies revealed that cerebral ischemia induced the expression of genes associated with a number of general functions including apoptosis, inflammation and metabolism. The identification of these specific cellular and molecular pathways has led to their implication in the progression of neuronal injury following stroke (Dirnagl et al., 1999, Iadecola and Alexander, 2001, Lo et al., 2003).

There are two major acute in vivo focal stroke models used to study the mechanisms involved in ischemic stroke (reviewed in Longa et al., 1989, McBean and Kelly, 1998, Mhairi Macrae, 1992, Yanamoto et al., 2003). In the permanent middle cerebral artery occlusion model (pMCAO), the MCA is blocked for a fixed period of time until the animal is sacrificed. Transient (ischemia–reperfusion) MCAO (tMCAO) is generated by occluding the MCA for a limited time period. The occlusion is then removed and blood flow is restored (reperfusion) to the ischemic brain. Although it is known that reperfusion improves outcome following ischemia, mechanisms associated with reperfusion can produce neuronal injury that contributes to infarct progression. Reperfusion injury has been shown to involve free radical formation, inflammation and expression of adhesion molecules (Barone and Feuerstein, 1999, del Zoppo et al., 2000, Iadecola and Alexander, 2001, Stoll et al., 1998, Traystman et al., 1991). There is disagreement in the field to which model is the most appropriate to represent human stroke. It has been suggested that the pMCAO model is more appropriate because blood flow is reduced and does not change much during the first few hours following stroke (STAIR, 1999). Also, reperfusion resulting from endogenous thrombolytic mechanisms occurs days rather than hours after stroke so this model may not be as relevant to the normal human condition. However, with the new thrombolytic therapies now available, the tMCAO model has become more clinically significant. Thrombolytic therapy is currently used to acutely restore blood flow to the brain following ischemia and has been shown to be effective in the treatment of ischemic stroke. Therefore, it is necessary to understand the distinct mechanisms that contribute to neuronal death induced by ischemia (occlusion) and reperfusion in order to determine appropriate therapeutic strategies.

In this study, we compare and contrast gene expression profiles in rat tMCAO and pMCAO stroke models using a new, powerful pathway data analysis tool, Expression Analysis Systematic Explorer (EASE). EASE is a bioinformatics program that provides statistical methods that facilitates the biological interpretation of gene lists derived from results of a microarray analysis (Hawse et al., 2003, Hawse et al., 2004, Hawse et al., 2005, Hosack et al., 2003, Warrenfeltz et al., 2004, Zeng et al., 2004). Recently, our laboratory used EASE analysis and showed that the neuroprotective effect of neuregulin-1 following tMCAO involved the attenuation of pro-inflammatory and oxidative stress related genes induced following ischemia (Xu et al., 2004). In this study, EASE analysis of the regulated genes indicated molecular functions and biological processes unique to each stroke model. These finding may provide novel views regarding the molecular mechanisms involved in stroke and lead to new neuroprotective strategies.