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Title 1

Unraveling the Ischemic Brain Transcriptome in a Permanent Middle 2

Cerebral Artery Occlusion Model by DNA Microarray Analysis 3

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Running title: Ischemic mouse brain transcriptome 5

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Authors 7

Motohide Hori1,2, Tomoya Nakamachi2,3, Randeep Rakwal2,4,5, Junko Shibato2, Keisuke 8

Nakamura2, Yoshihiro Wada2, Daisuke Tsuchikawa2, Akira Yoshikawa2, Keiji Tamaki1, and 9

Seiji Shioda2,* 10

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1Department of Forensic Medicine and Molecular Pathology, School of Medicine, Kyoto 12

University, Kyoto 606-8315, Japan 13

2Department of Anatomy I, School of Medicine, Showa University, 1-5-8 Hatanodai, 14

Shinagawa, Tokyo 142-8555, Japan 15

3Department of Center for Biotechnology, Showa University, Tokyo, Japan 16

4Anti-aging Medicine Funded Research Laboratories, Showa University, Tokyo, Japan 17

5Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 18

305-8572, Japan 19

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*Author for correspondence (shioda@med.showa-u.ac.jp); all submission and additional21

correspondence to Dr. Randeep Rakwal (e-mail- plantproteomics@gmail.com) 22

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SUMMARY 25

Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient 26

blood flow to the brain to meet metabolic demand, leading to tissue death (cerebral 27

infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the 28

protective effects of neuropeptides for alleviating brain ischemia, as well as the underlying 29

mechanisms of their action. The present study was initiated to investigate molecular 30

responses at the level of gene expression in ischemic brain tissue. To achieve this, we used a 31

mouse permanent middle cerebral artery occlusion (PMCAO) model in combination with 32

high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, the 33

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.008276Access the most recent version at DMM Advance Online Articles. Published 20 October 2011 as doi: 10.1242/dmm.008276

http://dmm.biologists.org/lookup/doi/10.1242/dmm.008276Access the most recent version at First posted online on 20 October 2011 as 10.1242/dmm.008276

2

right (ipsilateral) and left (contralateral) hemispheres of PMCAO model mice were dissected 34

at two time points, 6 and 24 hours post-ischemia. Total RNA from the ischemic (ipsilateral) 35

hemisphere was subjected to DNA microarray analysis on a mouse whole genome 4x44K 36

DNA chip using a dye-swap approach. Functional categorization using the gene ontology 37

(GO, MGD/AMIGO) of numerous changed genes revealed expression pattern changes in the 38

major categories of cellular process, biological regulation, regulation of biological process, 39

metabolic process, and response to stimulus. RT-PCR analysis on randomly selected highly 40

up- and down-regulated genes validated, in general, the microarray data. Using two time-41

points for this analysis, major and minor trends in gene expression/functions were observed 42

in relation to early- and late-response genes and differentially regulated genes that were 43

further classified into specific pathways or disease states. We also examined the expression of 44

these genes in the contralateral hemisphere, which suggested the presence of bilateral effects 45

and/or differential regulation. This study provides the first ischemia-related transcriptome 46

analysis of the mouse brain, laying a strong foundation for studies designed to elucidate the 47

mechanisms regulating ischemia and to explore the neuroprotective effects of agents such as 48

target neuropeptides. 49

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INTRODUCTION 51

Brain ischemia, also known as cerebral ischemia/ischemic stroke, is the third most common 52

cause of death after heart attack and cancer, resulting in major negative social and economic 53

consequences. Ischemic stroke, which results from cardiac arrest, cerebral arterial occlusion, 54

or severe vasospasm after subarachnoid ischemia, causes devastating damage to the brain and 55

represents a serious global health problem. Briefly, brain ischemia is a condition in which 56

there is insufficient blood flow to meet metabolic demands. It is known that an interruption of 57

blood flow to the brain for more than ten seconds results in a loss of consciousness, leading to 58

ischemia and irreversible brain damage. The most common cause of stroke is the sudden 59

occlusion of a blood vessel by a thrombus or embolism, resulting in an almost immediate loss 60

of oxygen and glucose to the cerebral tissue. Ischemia can be classified as either focal or 61

global. Focal ischemia is confined to a specific lesion whereas global ischemia emcompasses 62

a wide area of the brain (for further reading on the subject, see Gusev and Skvortsova, 2003). 63

Given the clinical importance of ischemia, it is not surprising that its causes, diagnosis, 64

and treatment are the focus of a major international research effort (see, for example, 65

Liebeskind, 2008; Slemmer et al., 2008; Dogrukol-Ak et al., 2009; Indraswari et al., 2009; 66

Kim et al., 2009; Chauveau et al., 2010; Gupta et al., 2010; Henninger et al. 2010; Rymner et 67

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al., 2010; Cucchiara and Kasner, 2010; Yunari and Hemmen, 2011; Fisher, 2011; Kunst and 68

Schaefer, 2011; Leiva-Salinas et al., 2011; Molina, 2011; Ramos-Fernandez et al., 2011; 69

Turner and Adamson, 2011; Wechsler, 2011). To provide an idea of the volume of research 70

carried out in this area, a keyword search on May 16th, 2011 using the PubMed National 71

Center for Biotechnology Information (NCBI) search engine revealed 78103 articles 72

containing the search term “brain ischemia”. More specifically, there were 58357 articles 73

containing “brain ischemia” AND “human” and 28253 for “brain ischemia” AND “animal”, 74

while “brain ischemia” AND “rat” and “brain ischemia” AND “mouse” revealed 9109 and 75

3912 articles, respectively. The subject has also been widely reviewed, with a PubMed search 76

using the keywords “brain ischemia” AND “review” resulting in a total of 9478 articles. In 77

order to understand the pathology of stroke, targeted gene expression and proteomics studies 78

have been carried out to investigate the role of particular genes and proteins. Decreased blood 79

flow during ischemia activates the synthesis of various genes and proteins which regulate the 80

ischemic process and/or are involved in the broader cellular response, depending on the 81

extent of the injury. Among these molecular factors we are also likely to find potentially 82

protective genes/proteins. 83

Our group has been working on ischemic models since 1994 (Mizushima et al., 1994; 84

Shimazu et al., 1994), with the goal of understanding the mechanisms underlying ischemia 85

and identifying molecular targets for its prevention/amelioration, including the use of 86

neuropeptides such as pituitary adenylate cyclase-activating polypeptide, PACAP. This has 87

led to the successful characterization of certain molecular components involved in cerebral 88

ischemia and the establishment of PACAP as a potential therapeutic agent for its prevention 89

(Shioda et al., 1998; Ohtaki et al., 2003, 2006, 2007, 2008a, b; Nakamachi et al., 2005, 2010). 90

The critical issue now is to elucidate exactly how any identified neuroprotective molecules 91

exert their effects. 92

Over the last decade, and particularly within the last 3 years, researchers have begun 93

focusing their attention on genomic approaches to investigate brain ischemia and 94

physiological responses to it (Jin et al., 2001; Büttner et al., 2008; Meschia, 2008; Juul et al., 95

2009; Haramati, 2009; Nakajima, 2009; Popa-Yao et al., 2009; Wagner, 2009; Di Pietro et al., 96

2009; Pruissen et al., 2009; Stamova et al., 2010; Zhan et al., 2010). These studies have 97

shown the importance of high-throughput transcript profiling like the DNA microarray 98

technique (DeRisi et al., 1997) for gaining insight into the ischemic brain. In the present study, 99

we have taken a different approach to the problem, and have adopted a whole genome DNA 100

microarray analysis approach (Masuo et al., 2011b, and references therein) as a means of 101

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providing an inventory of the time-dependent changes in global gene expression that occur at 102

the mRNA level in mice in response to ischemia. To achieve this, we have utilized a mouse 103

model of permanent middle cerebral artery occlusion (PMCAO) (Nakamachi et al., 2005, 104

2010) (see Supplementary Fig. 1a-g). Briefly, total RNA was extracted at 6 and 24 hours 105

from very fine powders of post-ischemic brain tissue, then subjected to DNA microarray 106

analysis using a mouse whole genome DNA chip (4x44K) with a dye swap approach, 107

followed by identification of changes in gene expression. Our results revealed a large number 108

of highly up- and down-regulated genes, whose involvement in the progression of and 109

response to ischemia is discussed in this paper. 110

111

RESULTS AND DISCUSSION 112

Permanent middle cerebral artery occlusion (PMCAO)-generated ischemia model mice 113

PMCAO (Supplementary Fig. 1b, d) was performed on 13 male mice (C57/BL6J), and the 114

results of the procedure were monitored by dissecting whole brains and visualizing the extent 115

of ischemia. Eleven mice (84.6%) survived the PMCAO procedure (data not shown). In 116

addition, a total of 10 sham (control) mice were used. We also injected 0.9% saline 117

intracerebroventrically in both the sham control and PMCAO model mice, consistent with 118

our long term goal of determining whether various neuropeptides can reverse the effects of 119

ischemia. Injection of saline was to the left hemisphere (contralateral; Supplementary Fig. 120

1c). The ischemic region was visualized by 2% 2, 3, 5-triphenyltetrazolium chloride (TTC; 121

Wako, Tokyo, Japan) in some mice to confirm the ischemic brain damage (Supplementary 122

Fig. 1e). Moreover, we also checked for neurological deficiency 24 hours after PMCAO, 123

using a routinely used methodology in our laboratory (Ohtaki et al., 2006; Dogrukol-Ak et al., 124

2009). Results showed neurological deficiency in 85% of these animals (PMCAO mice); 125

these were used for dissection of the brain tissues. After perfecting the technique, mice were 126

divided into 4 groups of 3 mice each for the control and PMCAO cohorts, and whole brains 127

were dissected following 6 or 24 hours of ischemia. The ipsilateral (right hemisphere, non-128

injected) and contralateral (left hemisphere; injected with saline) hemispheres without 129

olfactory bulb (OB) and cerebellum (Supplementary Fig. 1f, g) were quickly separated, 130

placed in 2.0 mL Eppendorf tubes, and deep frozen in liquid nitrogen. 131

132

Overview of the brain genomic response to ischemia 133

Quality of total RNA and level of GAPDH and-actin genes in the brain hemispheres 134

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To investigate global changes in gene expression in ischemic hemispheres, we first optimized 135

a protocol for total RNA extraction (described in section 2.4, Fig. 1, and Supplementary 136

Figs. 2 and 3). Here we would like to clarify that we refer to the ischemic brain hemisphere 137

(ipsilateral) as consisting of the infarct core, penumbra, and non-ischemic region under the 138

present experimental design and sampling of the brain tissue thereof. This provides an overall 139

picture of the ischemic brain hemisphere rather than one specific ischemic region. 140

Nevertheless, in the future it would be interesting to examine specific ischemic regions and 141

compare them with non-ischemic regions in the same hemisphere. The quantity and quality of 142

the total RNA, a critical factor in further downstream analyses, was confirmed, and this RNA 143

was then used for synthesizing cDNA. Prior to DNA microarray analysis, we examined the 144

expression of two commonly used house-keeping genes, namely glyceraldehyde 3-phosphate 145

dehydrogenase (GAPDH) and -actin (Supplementary Table 1) in both hemispheres at 6 146

and 24 hours. This allowed us to subsequently use these two genes as positive controls rather 147

than simply loading or using internal controls (for further reading, see Suzuki et al., 2000). 148

This simple test of gene expression showed that the mRNAs for GAPDH and -actin were 149

expressed almost uniformly across conditions (Supplementary Fig. 4). Following this 150

preliminary analysis of sample quantity and quality, we proceeded to conduct a DNA 151

microarray analysis using the ischemic (ipsilateral) brain hemisphere. 152

153

Changes in gene expressions in the ischemic brain and their functional categorization 154

Using the total RNA in the ischemic hemisphere and the 4x44K mouse whole genome DNA 155

microarray chip in conjunction with a dye-swap approach (as explained in the Materials and 156

Methods) genome-wide global gene expression profiles were obtained for ischemia-related 157

genes. The results revealed 1237 and 2759 cases of gene induction (> 1.5-fold) as compared 158

to 620 and 2102 cases of gene suppression (<0.75-fold) at 6 and 24 hours after PMCAO, 159

respectively (Fig. 2). These genes are shown in Supplementary Tables 2-5. For detailed 160

information on the changed gene expression profiles, readers are referred to the total gene 161

expression data files at the NCBI GEO data (GSE 28201) repository, submitted as part of this 162

publication. Interestingly, the majority of these changes were unique to either the 6 or 24 hour 163

time-points, reflecting the progression of ischemia with time. Nevertheless, 792 and 167 164

genes were found to be commonly up-regulated or down-regulated, respectively at 6 and 24 165

hours (Fig. 2, and Supplementary Tables 6 and 7). 166

We next categorized the affected genes based on their function under gene ontology 167

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(GO) terms of biological process (3440 and 6826 induced genes, and 1398 and 3531 168

suppressed genes at 6 and 24 hours, respectively), molecular function (875 and 1732 induced 169

genes, and 394 and 1021 suppressed genes at 6 and 24 hours, respectively), and cellular 170

component (1788 and 3522 induced genes, and 762 and 2015 suppressed genes at 6 and 24 171

hours, respectively). The functionally categorized genes are presented graphically in Fig. 3. It 172

should be noted that the same gene was sometimes included in different functional categories, 173

thus making the total numbers appear inflated. In the major category of biological process, 174

the sub-category of cellular process showed the largest number of gene changes (induction 175

and suppression), followed by biological regulation, regulation of biological process, and 176

metabolic process. In the second major category of molecular function, the binding activity 177

sub-category included the most cases of changed gene expression, followed by catalytic 178

activity, and molecular transducer activity. In the third major category of cellular component, 179

the major gene changes were in the cell, cell part, followed by the organelle, and extracellular 180

part sub-categories. From these data, it can be seen that, in general, the change in the pattern 181

of gene expression was time-dependent, i.e. the number of instances of increased and 182

decreased gene expression was higher at 24 hours than at 6 hours. This is in line with the fact 183

that cellular death progresses with time, i.e. following PMCAO the volume of ischemic brain 184

tissue increases to cover almost the entire right hemisphere by 24 hours. 185

186

Changes in gene expressions reveal differential time-dependent patterns 187

In the ipsilateral hemisphere and at the two time-points investigated in this analysis, major 188

and minor trends were observed in relation to gene expression/function in response to 189

ischemia. In a major trend, we were able to identify genes that were dramatically up-regulated 190

or down-regulated at 6 and 24 hours; these can be considered as early (pre-ischemia)- and late 191

(post-ischemia)-responsive genes. However, it must be emphasized that instances of up-192

regulation were more dramatic in the ischemic brain, indicating that ischemia increases gene 193

induction rather than gene supression. This is understandable given the fact that ischemic 194

damage increases with time, leading to irreversible apoptosis or cell death. For example, 195

S100a5 was the most highly up-regulated gene (41.25-fold) at 6 hours but at 24 hours showed 196

only a 12.97-fold induction, whereas Mmp8 and Mmp3 were increased 128.73- and 115.72-197

fold, respectively, at 24 hours but only showed a 12.44- and 6.23-fold induction respectively 198

at 6 hours. This trend differentiates early (S100a5)- and late (Mmp8/3)-responsive gene 199

expression in the ischemic brain. All the gene expression data are included in Supplementary 200

Tables 2-7 (see also Fig. 5). 201

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We further utilized the pathway- or specific disease states-focused gene classifications 202

available on the QIAGEN website (SABiosciences; www.sabiosciences.com) to reveal the 203

trend of predominant pathways affected in the ipsilateral (ischemic) hemisphere (Fig. 4). In 204

general, it can be seen that most of the categories show an increasing trend of gene up- or 205

down-regulation with time. The up- and down-regulated genes in each functional 206

classification at 6 and 24 hours after ischemia are listed in Supplementary Table 8. However, 207

the trends of gene quantity (numbers) and quality (function) in the respective pathways varied 208

between the two time points studied. This can be expected as the ischemic area progresses 209

with time in the ipsilateral hemisphere. For example, genes related to endothelial cell biology, 210

inflammatory response and autoimmunity pathways, and atherosclerosis showed strongly 211

increased expression at 24 hours. On the other hand, DNA repair genes were only seen at the 212

24 hour but not the 6 hour time point. Some of these gene expression trends are discussed 213

below in relation to ischemia. 214

S100 calcium-binding protein family 215

The S100 protein family constitutes the largest group of Ca2+-binding proteins of the EF-hand 216

type involved in cation homeostasis (Schafer and Heizmann, 1996; Heizmann and Cox, 1998). 217

We found that the mRNA level corresponding to the S100a5 gene is increased in the ischemic 218

brain. S100a5 is known as an unusual member of the S100 protein family in that it has a high 219

affinity for Ca2+, Zn2+, and Cu2+. S100a5 immunoreactivity was found in neuronal dendrites, 220

but not in neuronal soma or any glial cells in the rat brain (Schäfer et al., 2000). To the best of 221

our knowledge, the function of S100a5 in the ischemic brain is not known, but another family 222

member, S100b, has been shown to exhibit Cu2+ affinity and suppress the hemolysis of mouse 223

erythrocytes induced by CuCl2. Furthermore, when Escherichia coli are transformed with a 224

vector containing S100b cDNA, cell damage as a result of copper-induced oxidative stress is 225

also reduced (Nishikawa et al., 1997). It is tempting to speculate that S100a5 may also exert 226

such a protective role. It should be noted, however, that this gene was previously identified as 227

a novel hypoxia-inducible gene based on its up-regulation after 6 hours of hypoxia (Tang et 228

al., 2006). S100a5 may protect neurons against acute ischemic damage. Around ten S100 229

family members were reported and reviewed in 2005 with respect to nervous system function 230

and neurological disorders (Zimmer et al., 2005). Since then six more members of this family 231

have been identified. In our analysis, we also identified changes in the expression of other 232

members of the S100 family, namely S100a9 and S100a8 (up-regulated at 6 and 24 hours), 233

and S100a11, S100a14, S100a4, S100a6, S100a3, S100a15, and S100a10 (up-regulated only 234

at 24 hours). The early and late up-regulation of these genes suggests their involvement in 235

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both signal transduction processes and the inflammatory response. 236

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Matrix metalloproteinases 238

Two genes belonging to the matrix metalloproteinase (MMP) enzyme family, namely Mmp8 239

and Mmp3, showed the highest level of up-regulation in response to ischemia. As the name 240

suggests, these enzymes are known to participate in the degradation of components of the 241

extracellular matrix. The MMPs constitute a family of more than 20 endopeptidases (Yong et 242

al., 2001; Crocker et al., 2004; Cauwe et al., 2007). Although the induction and role of Mmp8 243

in ischemia has not previously been reported, this gene has been implicated in inflammatory 244

arthritis (Cox et al., 2010), lung injury (Albaiceta et al., 2010), atherosclerosis (Laxton et al., 245

2009), and periodontitis (Kuula et al., 2009). Taken together with the published reports of 246

Mmp8 function, our finding suggests a protective role for Mmp8 in ischemic tissues. This 247

could occur via the processing of anti-inflammatory cytokines and chemokines by MMP8. As 248

MMP8 is a proteinase, it may cleave and release certain signaling factors in the extracellular 249

matrix very early stage after ischemia (6 hours), and then subsequently contribute to the 250

activation of defensive responses at a semi-acute phase (24 hours). MMP8 is also known as 251

neutrophil collagenase and is released from activated neutrophils; this suggests that the 252

activated neutrophil starts invasion or migration processes (Dejonckheere et al., 2011). 253

On the other hand, the Mmp3 gene has been associated with the pathogenesis of a 254

number of diseases, including stroke, brain trauma, and neuroinflammation (Rosenberg, 255

2002). The group of Rosenberg et al (2001) also previously reported MMP3 and MMP9 256

immunoreactivity at 24 hours after ischemia; MMP3 and MMP9 were found to co-localize 257

with activated microglia and ischemic neurons. It has been previously reported that a small 258

amount of MMP-3 is needed to activate proMMP-9 in endothelial cells, amplifying changes 259

in brain blood vessel properties (Rosenberg et al., 2001). Recently, an important study 260

regarding the role of MMP3 inside the cell was published, in which it was shown that the 261

catalytically active, cleaved form of MMP3 (actMMP3) is produced intracellularly in 262

oxidatively stressed neurons and plays a role in apoptotic signaling (Choi et al., 2008). The 263

same group further demonstrated the role of MMP3 in neuronal apoptotic signaling 264

downstream of caspase12 during endoplasmic reticulum stress (Kim et al., 2010). MMP3 may 265

act as an apoptosis-inducing factor as well as a protease in the ischemic brain. Interestingly, 266

our analysis also identified a further five Mmp gene family members, including, Mmp9, 267

Mmp12, Mmp13, Mmp14, and Mmp19. The MMP9 protein has been reported to increase after 268

cerebral focal ischemia in rats (Romanic et al., 1998). We also found a 4.28-fold increase in 269

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Mmp9 expression at 24 hours after ischemia, which is in the line with the observation of 270

MMP9 protein expression. It has been recently reported that MMP9 released from bone 271

marrow-derived cells contributes to disruption of the blood-brain barrier that causes brain 272

edema in stroke (Wang et al., 2009). Thus, the up-regulation of MMP9 may correlate with 273

brain edema formation. 274

In all, these results suggest another novel finding indicating the involvement of 275

multiple MMPs in the ischemic brain. However, given the high levels of induction of Mmp8 276

and Mmp3, we hypothesize a particularly important role for these two family members in 277

ischemia. 278

279

Chemokines 280

The chemokines are chemotactic cytokines which, together with their receptors expressed on 281

leukocytes, play critical roles in the extravasation and migration of leukocytes under 282

inflammatory conditions (Gerard and Rollins, 2001; Semple et al., 2010). Two classes of 283

chemokines have been identified based on their structure, namely CXC and CC. The C refers 284

to the two N-terminal cysteine residues, and the classes are defined depending on whether or 285

not there is an amino acid between them (i.e. c-x-c versus c-c) (Rossi and Zlotnik, 2000). In 286

addition, chemokines have been reported to be important players in stroke and its 287

pathogenesis (Semple et al., 2010; Brait et al., 2011). Brait et al. (2011), using a PCR array of 288

84 genes, recently reported the expression of chemokines in ischemic mouse brain at 4, 24 289

and 72 h after 0.5 h of cerebral ischemia. 290

Similarly, in our study we also identified changes in the expression of numerous 291

chemokine-related genes, including genes encoding for chemokine (c-x-c motif and c-c motif) 292

ligands that were highly up-regulated by ischemic stress. For example, expression of the 293

Cxcl1 gene was increased 13.25-fold and 69.79-fold at 6 and 24 hours respectively, whereas 294

the Cxcl3 gene was only up-regulated (117.79-fold) at 24 hours. These trends reveal the 295

differential and specific regulation of the Cxcl family members in the brain. Other than these 296

two genes, we also identified Cxcl2, Cxcl10, Cxcl4, and Cxcl7 as being up-regulated 297

following 6 and 24 hours of ischemia, whereas Cxcl11, Cxcl16, and Cxcl13 were found to be 298

up-regulated only at 24 hours. In the c-c motif class, we identified the Ccl4 gene as being up-299

regulated 17.80- and 44.63-fold at 6 and 24 hours. Ccl2, Ccl7, Ccl24, Ccl11, Ccl17, Ccl9, 300

Ccl5, Ccl12, and Ccl6 were also up-regulated at both time-points. However, the Ccl19 gene 301

was found to be up-regulated specifically at 24 hours. 302

In addition, the chemokine receptor genes Ccr2, Ccr7, and Ccr1 were induced at 24 303

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hours, whereas the chemokine receptor-like gene Ccrl2 was induced at both 6 and 24 hours. 304

Although there has been some discussion regarding the pharmacological inhibition of 305

chemokine ligands and receptors as a means of reducing post-ischemic neuronal damage and 306

cell death, no chemokine has yet emerged as the most valid target (see also Brait et al., 2011). 307

Nonetheless, our study provides information on numerous differentially expressed chemokine 308

genes whose further analysis might shed light on their role in the ischemic brain. 309

310

Interleukins 311

Another example of a highly induced gene was interleukin 6 (Il6), which is also known to be 312

an important inflammatory cytokine in ischemic brain tissue (Ohtaki et al., 2006). Expression 313

of this gene was 10.35-fold and 89.23-fold up-regulated at 6 and 24 hours, respectively. IL6, 314

which was identified as a B-cell stimulating factor (Kishimoto, 1989) belongs to a subfamily 315

of cytokines that include leukemia inhibitory and ciliary neurotrophic factors, and use gp130 316

as a common receptor subunit. IL6 has been previously shown to play a role in 317

neurodegeneration rather than neuroprotection in cerebral ischemia (Clark et al., 2000). 318

Another report also showed that the infarct volume after temporary MCAO was similar in 319

wild-type and knockout mice lacking IL6 (Pera et al., 2004). Endogenous IL6, which 320

transiently increases in the acute phase of cerebral ischemia, plays a critical role in preventing 321

damaged neurons from undergoing apoptosis; this role may be mediated by Stat3 activation. 322

IL6 signaling could be used as a new target for stroke therapy, but exogenous IL6 323

administration is unlikely to be effective for treating brain ischemia given that excessive IL6 324

sometimes generates harmful effects, such as inducing fever (Yamashita et al., 2005). Our 325

results are in line with previous reports showing a critical role for Il6 in brain ischemia 326

progression. 327

In contrast to IL6, IL1-delta (Mus musculus interleukin 1 family, member 5) 328

expression is dramatically suppressed after ischemia (0.49-fold and 0.20-fold down-regulated 329

at 6 and 24 hours, respectively). IL1-delta is a subtype of the IL1 ligand, which shows strong 330

homology to the IL1 receptor antagonist IL1ra (Debets et al., 2001). Although the function of 331

IL1-delta in the brain is still unclear, it was reported that IL-1-delta blocked IL1-epsilon 332

function which activates the transcription factor NF-kappa-B (NF-κB) through the orphan 333

receptor IL1Rrp2. The NF-κB signal is activated by inflammatory responses; some anti-334

inflammatory drugs, various non-steroidal anti-inflammatory drugs, and glucocorticoids are 335

potent inhibitors of NF-κB (D'Acquisto et al., 2002). NF-κB activation also contributes to the 336

progress of brain ischemia (Harari et al., 2010). In addition, IL1-delta mRNA was strongly 337

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down-regulated in TNF-α-treated human adipocytes (Do et al., 2006). IL1-delta may act as an 338

anti-inflammatory or cytoprotective factor in brain ischemia. 339

The DNA microarray analysis in the present study provided new information for the 340

involvement of numerous interleukin family members in brain ischemia. We identified 341

interleukin1 receptor, type II (Il1r2), Il8rb (β), Il1a (α), Il17rb (homolog short isoform 342

precursor), Il19, Il31ra, Il1b, Il4ra, Il11, and Il1r1 as being up-regulated at 6 and 24 hours, 343

whereas Il18rb, Il7r, and Il1rap (receptor accessory protein) were induced only at 6 hours. At 344

24 hours, the Il1rn (receptor antagonist), Il18rap, Il21, Il1f9 (family), Il12rb1, Il13ra1, Il2rg 345

(γ), Il1rl1 (receptor-like), Il28ra, Il12b, Il13ra2, Il5, and Il6ra genes were found to be 346

specifically up-regulated. These results again reflect the importance of global gene profiling 347

approaches to investigate the brain. 348

349

Confirmation of gene expression by RT-PCR and differential expression of their 350

mRNAs in the ipsilateral and contralateral hemispheres 351

For confirmation of alterations in gene expression by DNA microarray, we randomly selected 352

15 genes with annotated functions that were highly up- or down-regulated in the ischemic 353

brain (8 genes in each group, as listed in Supplementary Table 1). The mRNA expression 354

profiles obtained by RT-PCR (Fig. 5) revealed that the DNA microarray data could be 355

validated using appropriate primer design. In addition, we also examined how those genes 356

which displayed altered expression patterns in the ipsilateral (ischemic) hemisphere behaved 357

in the contralateral (non-ischemic) hemisphere. This was done i) to confirm that these genes 358

are indeed ischemia-related, and ii) to uncover any potential bilateral effects. We also 359

reasoned that this parallel analysis would reveal any effects of saline injection on gene 360

expression. 361

Up-regulated genes 362

RT-PCR analysis revealed that S100a5 mRNA was also expressed in the non-ischemic region 363

(lanes 6 and 8, Fig. 5A). This was a surprising finding which we are unable to fully explain. 364

Nevertheless, it is possible that the S100a5 gene was influenced bilaterally following 365

ischemia, i.e. its expression was modulated by events occurring in the ischemic hemisphere. 366

The other member of this family, S100a8, was primarily expressed in the ischemic 367

hemisphere, but not (or only minimally) expressed in the non-ischemic hemisphere, 368

suggesting that S100a5 and S100a8 show differential expression in the non-ischemic 369

hemisphere. In the case of the heat shock protein (HSP) 1a (Hsp1a) gene, a high level of 370

expression was confirmed in the ischemic hemisphere at 6 hours. However, this gene was also 371

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constitutively expressed, albeit at low levels in the brain, which is logical considering the 372

important role of HSPs in cellular processes. Interestingly, a recent study has shown that 373

astrocyte targeted overexpression of HSP72 (or SOD) can reduce neuronal vulnerability to 374

forebrain ischemia (Xu et al., 2010). RT-PCR analysis revealed that Mmp3 was more highly 375

expressed than Mmp8, although both genes showed a high level of expression at 24 hours in 376

the ischemic region. Surprisingly, Mmp3 and Mmp8 genes showed dramatically opposite 377

regulation in the non-ischemic region. Considering the relatively high level of expression of 378

Mmp3 in the contralateral hemisphere of the sham controls, the possibility that the increase in 379

its mRNA expression was a result of the saline injection cannot be ruled out. In a similar 380

fashion, the Cxcl1 gene was strongly induced in the ischemic region, but not in the non-381

ischemic tissue. The Mmp3, CxCl1, Ccl4, Il6 genes of sham controls in the contralateral 382

hemisphere were more highly expressed than on the ipsilateral side, suggesting that the up-383

regulation may be a result of wounding, i.e. by the intracerebroventricular saline injection. 384

385

Down-regulated genes 386

To date, no known brain function of the Smg6 (Smg6-homolog, nonsense mediated mRNA 387

decay factor, C. elegans) gene has been described. Our findings provide the first reported 388

expression data of this gene in the mouse brain and its down-regulation under ischemia. The 389

Gm711 gene is a probable protein kinase-like protein, the expression of which has again not 390

been previously reported in the brain. However, our results demonstrated that it is down-391

regulated following 24 hours of ischemia. Lama1, which encodes for laminin, an extracellular 392

matrix constituent similar to the MMPs, was prominently down-regulated in the ischemic 393

region. A previous report has described laminin degradation in the CA1 and CA2 areas of 394

C57BL/6 mice subjected to 20 minutes of global cerebral ischemia (Lee et al., 2009). 395

Furthermore, this degradation could be reduced by the use of the tetracycline antibiotic 396

doxycycline via the inhibition of MMP9. Tnfsf10, the tumor necrosis factor (ligand) 397

superfamily member 10 gene, which is constitutively expressed in the brain, was found to be 398

dramatically reduced in the ischemic region, and also to some extent in the non-ischemic 399

region. Also called the proapoptotic cytokine TNF-related apoptosis-inducing ligand or 400

TRAIL, its function in ischemia remains to be clarified. Expression of the Aff2 gene, which 401

has been implicated in neurodevelopmental diseases, was found to be reduced following 402

ischemia (Vogel and Gruss, 2006). The Ankk1 gene is a predicted kinase shown to be 403

expressed exclusively in astrocytes in the adult CNS in humans and rodents (Hoenicka et al., 404

2010). Although its function in relation to ischemia remains unknown, its mRNA was reduced 405

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in the ischemic region. Finally, the p2ry12 gene encodes the purinergic P2Y receptor, G-406

protein coupled 12 protein, with a role in the vessel wall response to arterial injury and 407

thrombosis. Down-regulation of this gene was seen in both ischemic and non-ischemic 408

regions. 409

410

CONCLUDING REMARKS 411

Our study provides the first inventory of ischemia-related and/or responsive genes in the 412

mouse brain. Based on a high-throughput transcriptomics approach on a 44K DNA 413

microarray chip, we have identified not only numerous genes with potential involvement in 414

the regulation of brain ischemia, but also most of the genes that have previously been 415

reported in targeted studies. For example, the most prominent gene with known involvement 416

in regulating ischemia identified here was Il6. Apart from IL6 and related interleukins, we 417

also found that the calcium-binding protein S100a and members of the matrix 418

metalloproteinase and chemokine gene families showed the most significant changes 419

following 6 and 24 hours of ischemia. Interestingly, many genes also showed differential 420

expression in the non-ischemic contralateral hemisphere, revealing the identity of specific 421

ischemia-related genes. Our study further highlights the usefulness of global gene expression 422

profiling in searching for changes in gene expression and delineating the molecular events in 423

a defined experimental model, in this case the ischemic brain. Elucidating the expression 424

pattern of each differentially expressed gene in specific brain regions and determining the 425

level of protein synthesis will be the next experimental step. These future studies will provide 426

information on the sites of gene expression and increase our understanding of the functional 427

role of various genes in response to an ischemic injury. Moreover, and consistent with the 428

long-term goals of our group, the identified gene candidates will inform our investigations of 429

the effect of target neuropeptide/s in potentially controlling/reversing ischemia. 430

431

METHODS 432

Animals and husbandry 433

C57BL/6J mice purchased from Charles River (Kanagawa, Japan) were used in this study. 434

Thirty male mice (9-weeks-old, 25 ~ 35 g body weight) were housed at the Animal Institution 435

in Showa University in acrylic cages (8 mice / cage) at 23ºC, and maintained with a standard 436

12 h light/dark cycle with optimum humidity and temperature control. All animals were given 437

access to tap water and laboratory chow ad libitum. All animal care and experimental 438

procedures were approved by the Institutional Animal Care and Use Committee of Showa 439

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University (School of Medicine), Tokyo, Japan. 440

441

Permanent middle cerebral artery occlusion (PMCAO) and sham control 442

To generate the PMCAO model, mice were anesthetized with 4% sevoflurane (induction) and 443

2% sevoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An 444

incision was then made in the cervical skin followed by opening of the salivary gland, and 445

visualization of the right common carotid artery. The external carotid artery was exposed 446

through a midline cervical incision. For PMCAO, we used the intraluminal filament technique, 447

whereby a 7-0 monofilament nylon suture with its tip slightly rounded by heating was 448

inserted into the common carotid artery, then positioned in the middle cerebral artery 449

(Supplementary Fig. 1d), after which the wound was sutured closed. In sham control 450

animals, the external carotid artery was exposed and then the wound was sutured. In both the 451

sham control and PMCAO cases, 1 µl of saline (0.9% NaCl) was injected 452

intracerebroventrically, and the animals were returned to their cages. A total of four groups 453

were prepared: two groups of six and seven mice in the PMCAO cohorts at 6 and 24 h after 454

operation, respectively, and five mice each in the control (sham) groups at 6 and 24 h after 455

operation, respectively. We used three mice each in PMCAO groups that exhibited 456

neurological grades G1 and G2 (Ohtaki et al., 2006) and three mice each at random in sham 457

groups for the subsequent downstream analysis. Some of the mice were examined for 458

ischemia by TTC staining of brain sections (2 mm slices) at 37ºC for 10 minutes (Ohtaki et al., 459

2006; Dogrukol-Ak et al., 2009) using some of the PMCAO mice brains (Supplementary 460

Fig. 1e). 461

462

Dissection of brain and storage of samples 463

Six or 24 hours post-injection of saline, the mice were removed from their cages, decapitated, 464

and their brains carefully removed on ice. The left (contralateral) and right (ipsilateral) 465

hemispheres were dissected and placed in 2 mL Eppendorf tubes, which were then quickly 466

immersed in liquid nitrogen before being stored in -80ºC prior to further analysis (Fig. 1a). 467

468

Grinding of the brains and total RNA extraction 469

The deep-frozen brain hemispheres were transferred to a pre-chilled (in liquid nitrogen) 470

mortar and pestle and ground to a very fine powder with liquid nitrogen. The scheme for 471

preparation of fine tissue powders for downstream gene analysis is given in Figure 1a (see 472

also Masuo et al., 2011a, b). The powdered samples were transferred to 2 mL Eppendorf 473

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microtubes and stored in aliquots at -80ºC until used for extraction of total RNA or protein. 474

Total RNA was extracted from ~60 mg sample powder using the QIAGEN RNeasy Mini Kit 475

(QIAGEN, Germantown, MD, USA). The total RNA extraction protocol is briefly illustrated 476

in (Fig. 1b; see also Supplementary Fig. 2, and for additional information Masuo et al., 477

2011b; Ogawa et al., 2011). To verify the quality of this RNA, the yield and purity were 478

determined spectrophotometrically (NanoDrop, Wilmington, DE, USA) and visually 479

confirmed using formaldehyde-agarose gel electrophoresis (Fig. 1b, and Supplementary Fig. 480

3). 481

482

cDNA synthesis and reverse transcription-polymerase chain reaction (RT-PCR) 483

To validate the total RNA quality and subsequently synthesized cDNA, RT-PCR was 484

performed. Two commonly used genes, namely GAPDH and -actin were used for RT-PCR. 485

The 3’-UTR gene-specific primers are shown in Supplementary Table 1. Briefly, total RNA 486

samples (from both the ipsilateral and contralateral hemispheres) were first DNase-treated 487

with RNase-free DNase (Stratagene, Agilent Technologies, La Jolla, CA, USA). First-strand 488

cDNA was then synthesized in a 20 μL reaction mixture with an AffinityScript QPCR cDNA 489

Synthesis Kit (Stratagene) according to the protocol provided by the manufacturer, using 1 μg 490

total RNA isolated from each control and treated brain sample. The reaction conditions were: 491

25ºC for 5 minutes, 42ºC for 5 minutes, 55ºC for 40 minutes and 95ºC for 5 minutes. The 492

synthesized cDNA was made up to a volume of 50 μL with sterile water supplied in the kit. 493

The reaction mixture contained 0.6 μL of the first-strand cDNA, 7 pmols of each primer set 494

and 6.0 μL of the Emerald Amp PCR Master Mix (2X premix) (TaKaRa Shuzo, Shiga, Japan) 495

in a total volume of 12 μL. Thermal-cycling (Applied Biosystems, Tokyo, Japan) parameters 496

were as follows: after an initial denaturation at 97ºC for 5 minutes, samples were subjected to 497

a cycling regime of 20 to 40 cycles at 95ºC for 45 s, 55ºC for 45 s, and 72ºC for 1 minute. At 498

the end of the final cycle, an additional extension step was carried out for 10 minutes at 72ºC. 499

After completion of the PCR the total reaction mixture was spun down and mixed (3 μL), 500

before being loaded into the wells of a 1.2/1.8% agarose [Agarose (fine powder) Cat no. 501

02468-95, Nacalai Tesque, Kyoto, Japan] gel. Electrophoresis was then performed for ~22 502

minutes at 100 Volts in 1X TAE buffer using a Mupid-ex electrophoresis system (ADVANCE, 503

Tokyo, Japan). The gels were stained (8 μL of 10 mg/mL ethidium bromide in 200 mL 1X 504

TAE buffer) for ~7 minutes and the stained bands were visualized using an UV-505

transilluminator (ATTO, Tokyo, Japan). The RT-PCR protocol used in this study is detailed in 506

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Supplementary Fig. 5. 507

508

DNA microarray analysis in the ipsilateral (right) hemisphere 509

A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, 510

Palo Alto, CA, USA) was used for global gene expression analysis using the ipsilateral 511

(ischemic) hemisphere. Total RNA (900 ng; 300 ng each replicate pooled together) was 512

labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear 513

Amplification Kit (Agilent). Fluorescently labeled targets of control (sham) as well as treated 514

(PMCAO) samples were hybridized to the same microarray slide with 60-mer probes (Fig. 515

1C). As illustrated in Figure 1C, in this experiment we compared the PMCAO mice to sham 516

controls, i.e. the ipsilateral brain region of the PMCAO mice was compared with the same 517

right hemisphere of the control mice (Fig. 1A). A flip labeling (dye-swap or reverse labeling 518

with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with 519

unequal incorporation of the two Cy dyes into cDNA (Rosenzweig et al., 2004; Altman 2005; 520

Martin-Magniette et al., 2005). Briefly, to select differentially expressed genes, we identified 521

genes that were up-regulated in chip 1 (Cy3/Cy5 label for control and treatment, respectively 522

at 6 hours) but down-regulated in chip 2 (Cy3/Cy5 label for treatment and control, 523

respectively at 6 hours), for the ipsilateral brain hemisphere. The same selection criteria were 524

applied for chips 3 and 4 (24 hours). The use of a dye-swap approach provides a more 525

stringent selection condition for changed gene expression profiling than the use of a simple 526

single/two-color approach (Hirano et al., 2007, 2008, 2009; Tano et al., 2010a,b; Ogawa et al., 527

2011). 528

Hybridization and wash processes were performed according to the manufacturer’s 529

instructions, and hybridized microarrays were scanned using an Agilent Microarray scanner 530

G2565BA. For the detection of significantly differentially expressed genes between control 531

and treated samples each slide image was processed by Agilent Feature Extraction software 532

(version 9.5.3.1). Briefly, i) this program measured Cy3 and Cy5 signal intensities of whole 533

probes; ii) dye-bias tends to be signal intensity dependent, and therefore the software selected 534

probes using a set by rank consistency filter for dye-normalization; iii) normalization was 535

performed by LOWESS (locally weighted linear regression) which calculates the log ratio of 536

dye-normalized Cy3- and Cy5-signals, as well as the final error of the log ratio; iv) the 537

significance (P) value based on the propagate error and universal error models; v) the 538

threshold of significance for differentially expressed genes was <0.01 (for the confidence that 539

the feature was not differentially expressed); and vi) erroneous data generated due to artifacts 540

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were eliminated before data analysis using the software. The outputs of microarray analysis 541

used in this study are available under the series number GSE 28201, at the NCBI Gene 542

Expression Omnibus (GEO) public functional genomics data repository 543

(http://www.ncbi.nlm.nih.gov/geo/info/linking.html). 544

To validate the microarray data (ipsilateral hemisphere) RT-PCR was also performed 545

on randomly up- and down-regulated genes using 3’-UTR specific gene primers. In parallel, 546

we also examined the gene expression profile in the contralateral (left) hemisphere by using 547

the same genes for RT-PCR analysis. 548

549

ACKNOWLEDGEMENTS 550

MH gratefully acknowledges the members of Professor Seiji Shioda’s laboratory (at the 551

Department of Anatomy I) for their support and encouragement during this study. Authors 552

also appreciate the help of Dr. Tetsuo Ogawa (Showa University School of Medicine) and Mr. 553

Gaku Tamura (computer programmer) for their help with development of an Excel program 554

to sort the list of gene expression changes into the pathway- and specific disease states-555

focused gene classifications. Finally, we appreciate the Editor at SCIENCEDIT 556

(www.sciencedit.com) for a final edit and rewriting some part of the text of this manuscript. 557

558

COMPETING INTERESTS 559

There are no competing interests. 560

561

AUTHOR CONTRIBUTIONS 562

MH, TN, KN, YW, DT, and AY designed and generated the PMCAO model mice. MH, RR, 563

and JS designed and performed the DNA microarray and related experiments, analyzed the 564

data and wrote the paper. MH, TN, RR, KT, and SS checked, revised and finalized the paper. 565

566

SUPPLEMENTARY MATERIAL 567

Supplementary material for this article has been submitted online as a separate pdf file. 568

569

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ischemic attacks in humans regulates gene expression in rat peripheral blood. J Cereb 826

Blood Flow Metab 30, 110-118. 827

828

Figure Legends 829

830

Fig. 1. DNA microarray analysis of the ischemic brain (ipsilateral hemisphere). A) 831

Mouse whole brain and ipsilateral and contralateral hemispheres. Brain tissues were ground 832

to a fine powder in liquid nitrogen and stored at -80ºC. B) Total RNA extraction from the 833

finely powdered brain tissues. Total RNA quality was confirmed by agarose-gel 834

electrophoresis. C) DNA microarray experimental design. For further details, see the 835

Materials and Methods section. 836

837

Fig. 2. Differentially expressed genes in the ipsilateral hemisphere at 6 and 24 hours. 838

The gene lists are presented in Supplementary Tables 2-7. 839

840

Fig. 3. Functional categorization of the differentially expressed genes based on Gene 841

Ontology (GO). 842

843

Fig. 4. Pathway and disease states focused gene classification. The up- and down-844

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regulated genes at 6 (A) and 24 (B) hours after ischemia (ipsilateral hemisphere) were 845

classified based on the available categories of more than 100 biological pathways or specific 846

disease states in the SABiosciences PCR array list (QIAGEN; www.sabiosciences.com) for 847

Mus musculus. The numbers in the Y-axis represent number of genes in each category which 848

are indicated on the X-axis. 849

850

Fig. 5. mRNA expression profiles of 16 differentially expressed genes. A) up-regulated 851

genes, and B) down-regulated genes. Gel images on top show the PCR product bands stained 852

with ethidium bromide; the band intensities are also presented graphically below for clarity. 853

Lane numbers 1 to 8 indicate sham control (lanes 1, 3, 5, and 7) and PMCAO (lanes 2, 4, 6, 854

and 8) treatment, respectively. RT-PCR was performed as described in section 2.5 (Materials 855

and Methods) and the primers are detailed in Supplementary Table 1. 856

857

858

TRANSLATIONAL IMPACT 859

Clinical Issues 860

The early diagnosis and treatment of patients who have suffered episodes of brain or 861

myocardial infarction is critical in order to avoid the effects of ischemia and thereby improve 862

survival outcomes. The obstruction of cerebral blood flow induces time-dependent 863

pathophysiological changes in the brain tissue, including tissue necrosis and patient death if 864

the situation is not resolved quickly. In ischemic brain disease, patients may suffer from 865

symptoms that include neurological (e.g. palsy, speech difficulties) and psychological (e.g. 866

decrease in memory skill, anxiety) disorders among many other possible problems that could 867

further affect the patient and decrease quality of life. The most radical therapy for brain 868

ischemia is by thrombolytic reperfusion of the occluded artery, which restores blood flow to 869

peripheral regions again. The most important aspect about brain ischemia therapy after onset 870

of the disease is for the patient to change to a more healthy lifestyle, to undergo long-term 871

rehabilitation to improve residual function, to have a healthy diet, and to undertake an 872

appropriate amount of exercise as well as rest. 873

874

Results 875

Using the whole genome mouse 4x44K DNA microarray system with the dye-swap approach, 876

our study has revealed a large number of gene expression changes in the mouse brain 877

(ischemic hemisphere) at both 6 (1237 up-regulated and 620 down-regulated) and 24 hours 878

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(2759 up-regulated and 2102 down-regulated) following the ischemia episode. Seven 879

hundred and ninety two (792) and 167 genes were found to be commonly up- or down-880

regulated 6 and 24 hours post-PMCAO, respectively. Comparative and time-dependent 881

analysis of the ischemic brain hemisphere with sham controls revealed genes at different 882

levels and states of expression. As expected, genes linked to immune and inflammatory 883

responses were expressed more strongly; however, numerous novel genes and pathways 884

could also be identified for the first time in the ischemic hemisphere, thus justifying our 885

investment in the genome-wide global gene expression analysis approach. 886

887

Implications and future directions 888

This study i) provides researchers with a powerful resource in terms of gene candidates that 889

are up- or down-regulated in the ischemic brain, and ii) allows us to differentiate early and 890

late gene expressions that are involved not only in regulating ischemia but also in its 891

progression. Our results also confirm the importance of this work in shedding light on 892

mechanisms underlying brain ischemia. The gene candidates identified here could be targeted 893

in experiments involving knock-out mice (e.g., Il6 KO) for uncovering gene regulatory 894

processes and elucidating their effects on ischemia. Some genes, such as Cxcl/Ccl for 895

example, could also used for specific experiments designed to test their potential in drug 896

therapy. Finally, this study forms the basis for future research to examine the influence of 897

selected neuropeptides to reverse the effects of ischemia and provide clues about the 898

molecular mechanisms underlying this process. It is critical that we continue our investment 899

in the global analysis of gene and subsequent protein expression in ischemic mouse models to 900

unravel the molecular basis underlying ischemia. 901

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Fig. 1Hori et al.

Ipsilateral hemisphere (right)

Contralateral hemisphere (left)

A B

Each hemisphere was

ground to fine powder

&

Each sample powder was

transferred to Eppendorf tube and stored at -80°C

An optimized total RNA extraction protocol from the BRAIN

~ 60 mg sample tissue powder

QIAzol lysis buffer (QIAGEN)-Chloroform extraction

Supernatant taken for EtOH (70%) precipitation

RNeasy Mini Spin Column (Pink, QIAGEN) for RNA trap

Washing with RW1 buffer + DNaseI treatment

Washing with RPE buffer

Elute RNA with RNase free water (30-50 μL)

Concentration & Quality Check (NanoDrop)

28S

18S

SHAM SHAMPMCAO PMCAO

SHAM SHAM

PMCAO PMCAO

C

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Fig. 2Hori et al.

6 h 24 h

Num

ber

of d

iffe

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ially

exp

ress

ed t

rans

crip

ts

UP

DOWN500

1500

2500

3500

0

500

1500

2500

3500>1.5<0.75fold

>2.0<0.5fold

>1.5<0.75fold

>2.0<0.5fold

>1.5<0.75fold

>2.0<0.5fold

6 & 24 hcommon

Time of Ischemia

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Fig. 3Hori et al.

0 200 400 600 800 1000

biological adhesion

biological regulation

carbohydrate utilization

carbon utilization

cell killing

cell proliferation

cell wall organization or biogenesis

cellular component biogenesis

cellular component organization

cellular process

death

developmental process

establishment of localization

growth

immune system process

localization

locomotion

metabolic process

multi-organism process

multicellular organismal process

negative regulation of biological process

nitrogen utilization

pigmentation

positive regulation of biological process

regulation of biological process

reproduction

reproductive process

response to stimulus

rhythmic process

signaling

signaling process

sugar utilization

viral reproduction

Biological Process0 200 400 600 800 1000

antioxidant activity

binding

catalytic activity

channel regulator activity

chemoattractant activity

chemorepellent activity

electron carrier activity

enzyme regulator activity

metallochaperone activity

molecular transducer activity

protein tag

structural molecule activity

transcription regulator activity

translation regulator activity

transporter activity

Molecular Function

0 200 400 600 800 1000 1200

cell

cell part

extracellular region

extracellular region part

macromolecular complex

membrane-enclosed lumen

organelle

organelle part

synapse

synapse part

virion

virion part

Cellular Component

6 h UP

24 h UP

6 h DOWN

24 h DOWN

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Dise

ase

Mod

els &

Mec

hani

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D

MM

Dise

ase

Mod

els &

Mec

hani

sms

Acce

pted

man

uscr

ipt

Dise

ase

Mod

els &

Mec

hani

sms

New Fig. 4A, Hori et al.

0

2

4

6

8

10

12

14

16

18

20

0

10

20

30

40

50

60

70

Am

ino

Aci

d M

etab

olis

m I

ID

NA

Rep

air

Tel

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Tel

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ase

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6 h DOWN6 h DOWN

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Dise

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New Fig. 4B, Hori et al.

0

5

10

15

20

25

30

35

40

0

20

40

60

80

100

120

Mito

chon

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kins

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d Im

mun

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eros

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/ P

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24 h UP24 h UP

24 h DOWN24 h DOWN

B

Dise

ase

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man

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ase

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els &

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hani

sms

Fig. 5AHori et al.

0

20

40

60

80

100

120

140

S100a50

10

20

30

40

50

60

70

80

Ccl4

0

10

20

30

40

50

60

70

80

Hspa1a0

10

20

30

40

50

60

70

Mmp8

0

30

60

90

120

150

Mmp30

20

40

60

80

100

Il6

0

10

20

30

40

50

60

70

80

Cxcl10

10

20

30

40

50

60

70

S100a8

Rel

ativ

e ab

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nce

of m

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expr

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1 2 3 4 5 6 7 8

6 h 6 h24 h 24 hIpsilateral ContralateralA

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Fig. 5BHori et al.

Rel

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of m

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1 2 3 4 5 6 7 8

6 h 6 h24 h 24 hIpsilateral Contralateral

1 2 3 4 5 6 7 8

6 h 6 h24 h 24 hIpsilateral Contralateral

0

10

20

30

40

50

60

70

Smg60

20

40

60

80

100

Gm711

0

10

20

30

40

50

60

70

80

Lama10

20

40

60

80

100

Tnfsf10

0

20

40

60

80

100

Aff20

10

20

30

40

50

Ankk1

0

10

20

30

40

50

P2ry12

B

Dise

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Mod

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Mec

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Mod

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