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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
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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
237
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
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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
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signaling process
sugar utilization
viral reproduction
Biological Process0 200 400 600 800 1000
antioxidant activity
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catalytic activity
channel regulator activity
chemoattractant activity
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protein tag
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transcription regulator activity
translation regulator activity
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Molecular Function
0 200 400 600 800 1000 1200
cell
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gene
sis
Th1
-Th2
-Th3
Hyp
oxia
Sig
nalin
g P
athw
ayE
pith
elia
l to
Mes
ench
ymal
Tra
nsiti
on (
EM
T)
Tol
l-Lik
e R
ecep
tor
Sig
nalin
g P
athw
ayN
FB
Sig
nalin
g P
athw
ayM
olec
ular
Tox
icol
ogy
Pat
hway
Find
erM
ultip
le S
cler
osis
Hem
atop
oiet
ic S
tem
Cel
ls a
nd H
emat
opoi
esis
Inna
te a
nd A
dapt
ive
Imm
une
Res
pons
eA
ther
oscl
eros
is In
flam
mat
ory
Res
pons
e an
d A
utoi
mm
unity
End
othe
lial C
ell B
iolo
gy
PI3
K-A
KT
Sig
nalin
gP
olyc
omb
& T
ritho
rax
Com
plex
esD
NA
Rep
air
Ant
ivira
l Res
pons
eIn
terf
eron
,
Res
pons
eT
elom
eres
& T
elom
eras
eM
itoch
ondr
ial E
nerg
y M
etab
olis
mFa
tty A
cid
Met
abol
ism
Insu
lin S
igna
ling
Pat
hway
DN
A D
amag
e S
igna
ling
Pat
hway
Stre
ss &
Tox
icity
Pat
hway
Find
erE
pith
elia
l to
Mes
ench
ymal
Tra
nsiti
on (E
MT
)E
pige
netic
Chr
omat
in R
emod
elin
g Fa
ctor
sN
ecro
sis
Nitr
ic O
xide
Sig
nalin
g P
athw
ayIn
flam
mas
omes
Unf
olde
d P
rote
in R
espo
nse
Hom
eobo
x (H
OX
) G
enes
MA
P K
inas
e S
igna
ling
Pat
hway
Adi
poge
nesi
sH
eat S
hock
Pro
tein
sC
ell C
ycle
Cel
l Sur
face
Mar
kers
Est
roge
n R
ecep
tor S
igna
ling
mT
OR
Sig
nalin
g D
rug
Met
abol
ism
: Pha
se I
ID
rug
Met
abol
ism
Stre
ss &
Tox
icity
Epi
gene
tic C
hrom
atin
Mod
ifica
tion
Enz
ymes
Tum
or N
ecro
sis
Fac
tor
(TN
F) L
igan
d an
d R
ecep
tor\
nA
min
o A
cid
Met
abol
ism
IH
ypox
ia S
igna
ling
Pat
hway
Stre
ss R
espo
nse
to C
ellu
lar
Dam
age
Stem
Cel
l Sig
nalin
gC
ell D
eath
Pat
hway
Find
erL
ipop
rote
in S
igna
ling
Oxi
dativ
e St
ress
and
Ant
ioxi
dant
Def
ense
Nep
hrot
oxic
ityC
ellu
lar
Sene
scen
ceA
ngio
gene
sis
VE
GF
Sig
nalin
gD
iabe
tes
Glu
cose
Met
abol
ism
Dru
g M
etab
olis
m: P
hase
I E
nzym
esH
epat
otox
icity
Hyp
erte
nsio
nT
GF
/ B
MP
Sig
nalin
g P
athw
ayE
xtra
cellu
lar M
atrix
and
Adh
esio
n M
olec
ules
Mes
ench
ymal
Ste
m C
ell
Inte
rfer
on (I
FN) a
nd R
ecep
tor
Tum
or M
etas
tasi
sA
min
o A
cid
Met
abol
ism
II
Tra
nscr
iptio
n Fa
ctor
sP
rimar
y C
ilia
Em
bryo
nic
Ste
m C
ells
Den
driti
c an
d A
ntig
en P
rese
ntin
g C
ell
Fibr
osis
Wou
nd H
ealin
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otch
Sig
nalin
g P
athw
ayC
ell L
inea
ge I
dent
ifica
tion
Sign
al T
rans
duct
ion
Pat
hway
Find
erU
biqu
itina
tion
(Ubi
quity
latio
n) P
athw
ayTe
rmin
al D
iffer
entia
tion
Mar
kers
Fibr
osis
Infla
mm
ator
y C
ytok
ines
& R
ecep
tors
Cel
l Mot
ility
Mito
chon
dria
Skel
etal
Mus
cle:
Myo
gene
sis
& M
yopa
thy
Dru
g T
rans
port
ers
Com
mon
Cyt
okin
esT
h17
for
Aut
oim
mun
ity &
Inf
lam
mat
ion
Pro
tein
Pho
spha
tase
sN
euro
scie
nce
Ion
Cha
nnel
s &
Tra
nspo
rter
sA
lzhe
imer
’s D
isea
seC
ardi
otox
icity
Apo
ptos
isA
utop
hagy
Endo
thel
ial C
ell B
iolo
gyP
olyc
omb
& T
ritho
rax
Tar
get G
enes
Infla
mm
ator
y R
espo
nse
and
Aut
oim
mun
ityT
Hel
per C
ell D
iffer
entia
tion
Stem
Cel
lN
FB
Sig
nalin
g P
athw
ayC
ytos
kele
ton
Reg
ulat
ors
p53
Sig
nalin
g P
athw
ayO
besi
tyC
hem
okin
es &
Rec
epto
rsG
lyco
syla
tion
Inna
te a
nd A
dapt
ive
Imm
une
Res
pons
eW
NT
Sig
nalin
g P
athw
ayT
oll-L
ike
Rec
epto
r Sig
nalin
g P
athw
ayH
edge
hog
Sig
nalin
g P
athw
ayM
olec
ular
Tox
icol
ogy
Pat
hway
Find
erSt
em C
ell T
rans
crip
tion
Fact
ors
Neu
roto
xici
tyN
euro
gene
sis a
nd N
eura
l Ste
m C
ells
Par
kins
on's
Dis
ease
T-c
ell A
nerg
y an
d Im
mun
e T
oler
ance
Ath
eros
cler
osis
T
Cel
l Act
ivat
ion
Ost
eoge
nesi
sE
GF
/ P
DG
F S
igna
ling
JAK
/ S
TA
T S
igna
ling
Pat
hway
Neu
rotr
ansm
itter
Rec
epto
rs a
nd R
egul
ator
sG
-Pro
tein
-Cou
pled
Rec
epto
r Sig
nalin
gN
ucle
ar R
ecep
tors
and
Cor
egul
ator
sG
row
th F
acto
rH
emat
opoi
etic
Ste
m C
ells
and
Hem
atop
oies
isT
h1-T
h2-T
h3M
ultip
le S
cler
osis
Neu
rotr
ophi
n an
d R
ecep
tors
Syna
ptic
Pla
stic
ityH
untin
gton
's D
isea
seG
Pro
tein
Cou
pled
Rec
epto
rs
24 h UP24 h UP
24 h DOWN24 h DOWN
B
Dise
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
Acce
pted
man
uscr
ipt
Dise
ase
Mod
els &
Mec
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
unda
nce
of m
RNA
expr
essi
on
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 ContralateralA
Dise
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
Acce
pted
man
uscr
ipt
Dise
ase
Mod
els &
Mec
hani
sms
Fig. 5BHori et al.
Rel
ativ
e ab
unda
nce
of m
RNA
expr
essi
on
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
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
D
MM
Dise
ase
Mod
els &
Mec
hani
sms
Acce
pted
man
uscr
ipt
Dise
ase
Mod
els &
Mec
hani
sms
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