A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

  • Nick Mitsios1Email author,

    Affiliated with

    • Mohamad Saka1Email author,

      Affiliated with

      • Jerzy Krupinski2,

        Affiliated with

        • Roberta Pennucci1,

          Affiliated with

          • Coral Sanfeliu3,

            Affiliated with

            • Qiuyu Wang1,

              Affiliated with

              • Francisco Rubio2,

                Affiliated with

                • John Gaffney1,

                  Affiliated with

                  • Pat Kumar1,

                    Affiliated with

                    • Shant Kumar4,

                      Affiliated with

                      • Matthew Sullivan1 and

                        Affiliated with

                        • Mark Slevin1Email author

                          Affiliated with

                          BMC Neuroscience20078:93

                          DOI: 10.1186/1471-2202-8-93

                          Received: 04 April 2007

                          Accepted: 12 November 2007

                          Published: 12 November 2007

                          Abstract

                          Background

                          Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain.

                          Results

                          Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11.

                          Conclusion

                          Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

                          Background

                          Ischaemic stroke results from obstruction of blood flow in a major cerebral vessel and leads to deregulation of genes whose expression promotes ischemic neuronal death and subsequent neurological dysfunction [1, 2]. Under ischemic conditions, energy metabolism fails, and severe reduction in mRNA and protein synthesis occurs in the ischemic core region. The tissue surrounding this area (peri-infarcted region) is able to maintain some functions, such as ionic homeostasis and can be partially salvaged by blood recirculation [3, 4].

                          The precise molecular mechanisms involved in ischemia-induced brain injury remain poorly understood. Limited knowledge of the molecular mechanisms involved in tissue regeneration has been gained from animal experiments using the middle cerebral artery occlusion (MCAO) model which replicates, in many aspects, the neuropathological changes following stroke in humans [5]. Although the contralateral side of the brain is not totally unaffected by ischemic damage, the collection of experimental and reference control tissue from the same animal is a better comparison than using sham-operated control in rats, while in human samples the only control tissue available is contralateral hemisphere. In addition, using contralateral tissue as a control and the direct comparison with stroke hemisphere provides the best model for validation as it removes inter-patient genetic variation and also minimises the differences in potential degradation between the target and reference mRNAs. This has been applied previously in both human [6, 7] and animal [810] studies. Rao et al. in particular observed very few differences in gene expression between sham and contralateral cortex at 24 h of reperfusion following MCAO in the rat [9].

                          Analysis of ischemic brain tissue with techniques capable of studying multiple transcripts simultaneously can identify gene expression changes previously not known to be implicated in ischemic pathophysiology and may lead to development of new targets for stroke therapy [11]. DNA microarray technology has been used to investigate the expression of thousands of genes in a single hybridization experiment. Several experimental studies have examined alteration of gene expression in the postischemic rat brain using microarray technology [810, 1218], while blood genomic profiling in human stroke have been investigated in recent pilot studies [19, 20] (Table 1). Critical comparison of gene expression profiles after stroke in humans with those in animal models may lead to a better understanding of the pathophysiology of brain ischaemia and allow an evaluation of the usefulness of animal models in stroke research.
                          Table 1

                          Previous studies employing microarray approaches to study stroke

                           

                          Soriano et al. 2000

                          Jin et al. 2001

                          Kim et al. 2002

                          Rao et al. 2002

                          Schmidt-Kastner et al. 2002

                          Tang et al. 2002

                          Roth et al. 2003

                          Kim et al. 2004

                          Lu et al. 2004

                          Moore et al. 2005

                          Ford et al. 2006

                          Tang et al. 2006

                          Vikman and Edvinsson 2006

                          Our data

                          Material used

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                          Rat brain tissue

                           

                          Rat brain tissue

                             

                          Model of ischemia

                          Permanent focal MCAO

                          Transient global MCAO

                          Permanent focal MCAO

                          Transient focal MCAO

                          Transient focal MCAO

                          Permanent focal MCAO

                          Permanent focal MCAO

                          Transient focal MCAO

                          Transient focal MCAO

                          Blood from ischemic stroke patients

                          Permanent and transient focal MCAO

                          Blood from ischemic stroke patients

                          Post-mortem brain tissue from 11 stroke patients

                          Post-mortem brain tissue from 12 stroke patients and permanent focal rat MCAO

                          No of genes

                          750

                          374

                          1176

                          1263

                          9044

                          ~8,000

                          ~13,000

                          5,000

                          1,322

                          ~19,000

                          8784

                          ~39,000

                          7458

                          1176

                          Time after ischemia

                          3 hours

                          4 hours

                          24 hours

                          72 hours

                          6 hours

                          6 hours

                          24 hours

                          5 hours

                          24 hours

                          1 hours

                          3 hours

                          6 hours

                          24 hours

                          3 hours

                          6 hours

                          12 hours

                          1 days

                          2 days

                          4 days

                          30 min

                          4 hours

                          8 hours

                          24 hours

                          3 days

                          7 days

                          As soon as possible after hospitalization

                          24 hours

                          3 hours

                          5 hours

                          24 hours

                          7–10 days (obtained 2–3 days post-mortem)

                          1 hour-21 days (rat) and 2–37 days (human, obtained by 6 hours post-mortem)

                          Cut-off values

                          2.0-fold

                          1.7-fold

                          2.0-fold

                          2.5-fold

                          1.7-fold

                          2.0-fold

                          3.0-fold

                          2.0-fold

                          2.0-fold

                          -

                          2.0-fold

                          1.5-fold

                          -

                          2.0-fold

                          Confirmation of results

                          In situ hybridization, western blotting

                          Western blotting, immuno-histochemistry

                          RT-PCR

                          Real-time PCR, antisense knockdown, western blotting, immuno-histochemistry

                          Microarray analysis only

                          Real-time RT-PCR

                          Cell culture, in situ hybridization, western blotting, immuno-fluorescence

                          Cell culture, northern blotting, RT-PCR, western blotting, immuno-histochemistry

                          Real-time RT-PCR

                          Real-time RT-PCR

                          Microarray analysis only

                          Microarray analysis only

                          Real-time PCR, immuno-histochemistry

                          Cell culture, RT-PCR, western blotting, immuno-histochemistry, immuno-fluorescence

                          Selected molecules

                          NGFI-C

                          ARC

                          GRB2

                          SMN1

                          IFN-IP

                          NDGAP-1

                          NPR

                          SOCS-3

                           

                          NARP

                          SPR

                          SPIN2C

                          ARG1

                          LBP

                          PC4

                          FAK

                          Synaptic proteins

                          CD14

                          CD36

                          FcGR2A

                          IFNGR1 caspase-1 a-catenin

                            

                          LY64

                          ELK3

                          POU3F4

                          RHOA

                          PAK1

                          MMP11

                          INI1

                          Until recently, gene expression profiling had not been applied to patients dying of ischemic stroke, in part because human brain autopsies are not regularly obtained. Although tissue obtained from brain autopsies is generally of lower quality than that of brain biopsies obtained from living patients, the majority of RNA transcripts and proteins in the human brain are reasonably stable (compared to other tissues such as blood and kidney) and degrade to only a minor degree following death, thus making autopsy tissue a useful source for the isolation of nucleic acids and proteins [21]. Previous studies evaluating the mRNA quality in human post-mortem brain tissue have demonstrated a minimal effect upon their overall relative stability and indicated that frozen brains up to 72 hours post-mortem can be efficiently analyzed [22]. In line with that, in previous human brain studies, tissue was obtained up to a maximum of 6 hours [7], 40 hours [23], 45 hours [24] and 69 [6] hours following death. Moreover, after comparing mRNA levels in autopsies and biopsies, Castensson et al. [25] found a general similarity in the levels between the two groups, and suggested that mRNA levels in brain autopsy samples can provide clues about the brain in vivo. Interestingly, Almeida et al. [26] found that, even if performed on degraded RNA, RT-PCR can be used to provide a reliable estimate of in vivo mRNA levels, maybe due to the similarities in the rates of degradation between the target and reference mRNAs. Recently, Vikman and Edvinsson [27] investigated the gene expression in human brain after ischaemia using samples 7–10 days post-stroke; however, they obtained their samples after a considerable delay of 2–3 days post-mortem and they focused mainly on mRNA expression of receptors.

                          To identify the genes whose expression was changed in the human brain following ischaemia, we investigated the dynamic changes in gene expression in brain samples (collected within 6 h of death) from patients with various times of survival (2–37 days; Table 2) following stroke and compared them with those at various time-points (1 hour – 21 days) following middle cerebral artery occlusion (MCAO) in rats. The Atlas 1.2 cDNA microarray was used to screen for differential expression of 1176 genes and significantly de-regulated genes were selected through image analysis. We further investigated whether the altered mRNA and protein levels of a subset of deregulated molecules in the postischemic brain could be reproduced in an in vitro model of neuronal and endothelial cell culture under conditions of oxygen-glucose deprivation (OGD). The findings confirmed previous studies reporting that parallel screening of gene expression can detect both previously documented and novel transcriptional features of the cerebral response to ischemia, and demonstrated significant differences in gene expression between human stroke and the animal model.
                          Table 2

                          Clinical Details of Patients

                          Patient no.

                          Age/sex

                          Survival after stroke

                          NIHSS on admission

                          Hypertensiona

                          Coronary artery disease

                          Atrial fibrillation

                          History of TIA/previous stroke

                          Hypercholesterolemiab

                          Smoking

                          Obesityc

                          Cause of death

                          Antiplatelets

                          Statinsd

                          RSA-be

                          1

                          63/F

                          2 days

                          26

                          Yes

                          No

                          No

                          No

                          No

                          No

                          No

                          Large ischemic stroke

                          No

                          No

                          Yes

                          2

                          84/M

                          3 days

                          21

                          Yes

                          Yes

                          No

                          No

                          No

                          Yes

                          No

                          Malignant stroke

                          No

                          No

                          No

                          3

                          68/M

                          3 days

                          24

                          Yes

                          Yes

                          No

                          No

                          Yes

                          No

                          No

                          Brain oedema

                          No

                          No

                          Yes

                          4

                          84/M

                          6 days

                          22

                          Yes

                          Yes

                          No

                          No

                          No

                          No

                          No

                          Cardiac failure

                          Yes

                          Yes

                          No

                          5

                          51/M

                          9 days

                          25

                          Yes

                          No

                          No

                          Yes

                          No

                          No

                          No

                          Respiratory infection

                          Yes

                          No

                          No

                          6

                          74/M

                          15 days

                          22

                          Yes

                          Yes

                          No

                          No

                          No

                          No

                          Yes

                          Heart attack

                          Yes

                          No

                          Yes

                          7

                          86/M

                          15 days

                          14

                          Yes

                          Yes

                          No

                          No

                          Yes

                          No

                          No

                          Urinary infection

                          Yes

                          No

                          No

                          8

                          58/M

                          17 days

                          16

                          Yes

                          Yes

                          No

                          No

                          Yes

                          Yes

                          No

                          Cardiac infarction

                          Yes

                          No

                          No

                          9

                          74/M

                          20 days

                          12

                          Yes

                          Yes

                          No

                          No

                          No

                          No

                          No

                          Bronchial aspiration

                          Yes

                          No

                          No

                          10

                          73/M

                          26 days

                          14

                          Yes

                          Yes

                          No

                          No

                          Yes

                          No

                          Yes

                          Respiratory infection

                          Yes

                          No

                          Yes

                          11

                          75/M

                          29 days

                          20

                          No

                          Yes

                          Yes

                          No

                          No

                          Yes

                          No

                          Septic shock

                          Yes

                          No

                          Yes

                          12

                          60/F

                          37 days

                          18

                          Yes

                          Yes

                          No

                          No

                          Yes

                          Yes

                          No

                          Pulmonary embolism

                          Yes

                          No

                          No

                          a Blood pressure greater than 135/85 mmHg.

                          b Serum total cholesterol levels greater than 5.2 mmol.

                          c Body mass index greater than 30.

                          d Patients who were on statins before the ischemic stroke.

                          e Patients taking either angiotensin converting enzyme inhibitors or angiotensin type I receptor antagonists.

                          M = male; F = female; NIHSS = NIH Stroke Scale; TIA = Transient Ischaemic Attack.

                          Results

                          cDNA microarray analysis

                          The expression of ischemia-related genes was determined by comparing the infarct-induced expression (combined samples from infarcted and peri-infarcted areas) to that in the contralateral hemisphere: 77, 92 and 15 genes were de-regulated in stroke-affected regions in the 3 patient survival groups respectively, while 9, 51, 48, 166, 253, 117 and 261 genes were altered at the 7 different time-points in the animal model compared to the controls (Figure 1). The combined number of differentially expressed transcripts in stroke patients represented 6.5%, 7.8% and 1.3% respectively in each survival group of the total number of the genes on the microarray. These findings compare with 0.8%, 4.3%, 4%, 14.1%, 21.5%, 10% and 22.2% of genes respectively at each time-point in rats.
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2202-8-93/MediaObjects/12868_2007_Article_391_Fig1_HTML.jpg
                          Figure 1

                          Statistical analysis of microarray data. Total number of genes and number of overlapping genes (between the two array sets) deregulated following stroke in human and rat (A). Scatter plots representing the data dispersion over two logarithmic scales for all time-points in human (B) and rat (C).

                          In total, 126 genes were deregulated after stroke in humans and 335 in the rat MCAO model. However, these data are not directly comparable since many transcripts in the human array were not present in the rat array and vice versa. Out of a total of 393 genes present in both arrays, 31, 49 and 5 showed deregulated expression in the 3 patient groups respectively, whilst 7, 27, 26, 62, 107, 34 and 107 genes were deregulated at each of the 7 time-points respectively following rat MCAO (Table 3, Figure 1). Of the 393 overlapping transcripts, the expression of 36 was changed only in the human study, compared with 184 that were altered only in the animal model, while only 41 deregulated genes were shared between the two studies. Interestingly, the mean fold changes in the human data were much higher than in the rat.
                          Table 3

                          Genes deregulated in both human and animal stroke microarrays

                           

                          Human

                          Rat

                          Human

                          Rat

                          Gene name

                          GenBank

                          SwissProt

                          GenBank

                          SwissProt

                          Max/min

                          Days

                          Max/min

                          Time

                          c-jun proto-oncogene

                          J04111

                          P05412

                          X17163

                          P17325

                          4.4-fold

                          9 – 20

                          3.5-fold

                          1 h – 24 h

                          Matrix metalloproteinase 11

                          X57766

                          P24347

                          U46034

                          P97568

                          3.2-fold

                          2 – 20

                          2.6-fold

                          3 days

                          Calcium/calmodulin-dependent kinase (CAMK1)

                          L41816

                          Q14012

                          L24907

                          Q63450

                          17.2-fold

                          2 – 20

                          0.05-fold

                          21 days

                             

                          L26288

                          Q63084

                              

                          LIM domain kinase 1

                          D26309

                          P53667

                          D31873

                          P53669

                          3.6-fold

                          2 – 20

                          2.4-fold

                          3 days

                                 

                          0.4-fold

                          21 days

                          T-Lymphocyte maturation-associated protein

                          M15800

                          P21145

                          U31367

                          Q64349

                          1.7-fold

                          2 – 6

                          0.2-fold

                          21 days

                          Retinoic Acid Receptor beta

                          M84820

                          P28702

                          M81766

                          P49743

                          2.0-fold

                          2 – 6

                          0.1-fold

                          21 days

                           

                          S54072

                          P28703

                                

                          Tyrosine Phosphatase 1B

                          M31724

                          P18031

                          M33962

                          P20417

                          3.4-fold

                          2 – 6

                          0.2-fold

                          21 days

                          Adenosine A1 Receptor

                          S56143

                          P30542

                          M64299

                          P25099

                          2.6-fold

                          2 – 6

                          5.2-fold

                          4 hrs

                          Growth arrest & DNA damage-inducible 153

                          S40706

                          P35638

                          U30186

                          Q62804

                          2.4-fold

                          2 – 6

                          2.1-fold

                          3 days

                           

                          S62138

                                 

                          Glutamate Decarboxylase 67

                          M81883

                          Q99259

                          M34445

                          P18088

                          5.6-fold

                          2 – 6

                          2.5-fold

                          21 days

                          Glutamate Decarboxylase 65

                          M81882

                          Q99259

                          M72422

                          Q05683

                          22.7-fold

                          2 – 20

                          2.2-fold

                          3 days

                          Neurotrophin 3

                          M37763

                          P20783

                          M34643

                          P18280

                          5.1-fold

                          2 – 37

                          2.2-fold

                          12 hrs

                          Inhibitor of DNA binding 2

                          M97796

                          Q02363

                          D10863

                          P41137

                          5.6-fold

                          2 – 20

                          0.4-fold

                          21 days

                          Neuropeptide Y

                          K01911

                          P01303

                          M20373

                          P07808

                          8.8-fold

                          2 – 20

                          0.04-fold

                          21 days

                          Glia Maturation Factor beta

                          M86492

                          P17774

                          Z11558

                          Q63228

                          7.6-fold

                          2 – 6

                          0.04-fold

                          21 days

                          High Mobility Group Protein 1

                          M23619

                          P17096

                          M64986

                          P27109

                          4.3-fold

                          2 – 37

                          3-fold

                          4 h – 3 d

                              

                          P27428

                            

                          0.3-fold

                          21 days

                          Early Growth Response Protein 1

                          X52541

                          P18146

                          M18416

                          P08154

                          4.4-fold

                          2 – 20

                          3.9-fold

                          1 h – 12 h

                           

                          M62829

                           

                          J04154

                             

                          0.2-fold

                          21 days

                          TAT-Binding Protein 1

                          M34079

                          P17980

                          U77918

                          P97638

                          3.8-fold

                          2 – 20

                          0.4-fold

                          21 days

                          Glutathione S-Transferase 1

                          J03746

                          P10620

                          J03752

                          P08011

                          17.5-fold

                          2 – 20

                          10.8-fold

                          24 h – 21 d

                          Fibroblast Growth Factor Receptor 1

                          M63887

                          Q02063

                          D12498

                          Q04589

                          10.1-fold

                          2 – 20

                          4-fold

                          4 h – 24 h

                           

                          M63888

                          Q02065

                                
                           

                          M63889

                                 

                          Interleukin 10

                          M57627

                          P22301

                          L02926

                          P29456

                          2.4-fold

                          2 – 20 26 – 37

                          6.4-fold

                          21 days

                              

                          Q63263

                          0.2-fold

                             

                          Heat Shock Protein 27

                          X54079

                          P04792

                          M86389

                          P42930

                          0.6-fold

                          2 – 20

                          15.2-fold

                          4 h – 24 h

                          Heat Shock Protein 70

                          M11717

                          P08107

                          Z27118

                          Q63718

                          0.6-fold

                          2 – 6

                          9.4-fold

                          1 h – 24 h

                            

                          P19790

                                

                          Thioredoxin Peroxidase 1

                          L19185

                          P32119

                          U06099

                          P35704

                          4.9-fold

                          2 – 20

                          3.9-fold

                          21 days

                           

                          X82321

                          P31945

                                

                          Platelet-Derived Growth Factor A

                          X06374

                          P04085

                          L06894

                          P28576

                          1.6-fold

                          2 – 6

                          0.5-fold

                          21 days

                          Matrix Metalloproteinase 14

                          X83535

                          Q92678

                          X83537

                          Q10739

                          6.9-fold

                          2 – 6

                          3.3-fold

                          24 h – 3 d

                          Kinase receptor TYRO3 Sky proto-oncogene

                          D17517

                          Q06418

                          D37880

                          P55146

                          3.1-fold

                          9 – 20

                          4.0-fold

                          24 h – 3 d

                                 

                          0.4-fold

                          21 days

                          CSF-1-Receptor

                          X03663

                          P07333

                          X61479

                          Q00495

                          89.2-fold

                          9 – 20

                          2.8-fold

                          3 days

                          Insulin-like Growth Factor Binding Protein 2

                          M35410

                          P18065

                          J04486

                          P12843

                          79.7-fold

                          9 – 20

                          2.1-fold

                          3 days

                          Mitogen activated kinase 1/2

                          M84489

                          P28482

                          M64300

                          P27703

                          48.6-fold

                          9 – 20

                          0.3-fold

                          21 days

                          Aquaporin 4

                          U34846

                          P55087

                          U14007

                          P47863

                          18-fold

                          9 – 20

                          3.2-fold

                          3 days

                          erbB2 proto-oncogene Neu proto-oncogene

                          M95667

                          P04626

                          X03362

                          P06494

                          11.2-fold

                          9 – 20

                          2.7-fold

                          12 hrs

                           

                          M11730

                          Q14256

                                

                          L-type calcium channel β3

                          U07139

                          P54284

                          M88751

                          P54287

                          10.3-fold

                          9 – 20

                          8.6-fold

                          21 days

                          Ras-related protein RAB3A

                          M28210

                          P20336

                          X06889

                          P05713

                          13.9-fold

                          9 – 20

                          0.3-fold

                          21 days

                          CAMK-II beta

                          U50358

                          Q13554

                          M16112

                          P08413

                          1.8-fold

                          9 – 20

                          0.3-fold

                          21 days

                          Growth Factor Receptor-Bound 2

                          L29511

                          Q63057

                          D49846

                          Q63057

                          19.9-fold

                          9 – 20

                          2.7-fold

                          3 days

                           

                          M96995

                          Q14450

                           

                          Q14450

                              

                          Signal Transducer & Activator of Transcription 3

                          L29277

                          P40763

                          X91810

                          P52631

                          0.4-fold

                          9 – 20

                          6.6-fold

                          4 h – 3 d

                                 

                          0.05-fold

                          21 days

                          Neuronatin

                          U25033

                          Q16517

                          U08290

                          Q62649

                          11.1-fold

                          9 – 20

                          0.4-fold

                          21 days

                              

                          Q62663

                              

                          Glutathione S-Transferase P

                          X08058

                          P09211

                          X02904

                          P04906

                          3.1-fold

                          9 – 20

                          0.1-fold

                          1 hr

                          Glucocorticoid-regulated serine/threonine kinase GSK

                          AJ000512

                          O00141

                          L01624

                          Q06226

                          0.6-fold

                          26 – 37

                          2.4-fold

                          3 days

                                 

                          0.05-fold

                          21 days

                          Glucose Transporter 1

                          K03195

                          P11166

                          M13979

                          P11167

                          0.6-fold

                          26 – 37

                          11.6-fold

                          4 h – 21 d

                          Amongst these genes we examined in more detail a small subset with no prior report of a role in stroke (PAK1, MMP11 and INI1). PAK1 was only induced in the human study although present in both microarray sets, MMP11 was induced in both cases, while INI1 was induced in the human but was not present in the rat microarray set. To confirm the microarray data, RT-PCR was carried out on selected deregulated genes. The temporal expression patterns of these genes following RT-PCR showed good agreement with the corresponding expression profiles obtained from the microarray analysis, supporting the validity of the data obtained from the microarrays. Using Western blotting and immunohistochemistry, PAK1, INI1 and MMP11 protein expression and localization was determined in the contralateral and ipsilateral brain areas of individual stroke patients and rats subjected to MCAO, and in HBMEC and HFN exposed to OGD and reperfusion.

                          Integrase Interactor 1 (INI1)

                          In agreement with the microarray data, RT-PCR demonstrated an increase in ini1 mRNA levels in peri-infarcted and infarcted areas of patients who survived between 2 and 6 days following stroke (Figure 2A). Analysis of INI1 protein expression in samples from individual stroke patients showed that protein levels were increased in peri-infarcted and infarcted regions in 8 of 12 samples (Table 4; Figure 2Bi and 2Bii). Only one patient who survived for 3 days after stroke showed decreased protein expression. Cells from contralateral white matter were not stained for INI1 but some weak neuronal cytoplasmic staining was seen in grey matter (Figure 2Ci). An increase in its expression was observed in the cytoplasm of cells with the morphological appearance of glia and microvessels from peri-infarcted and infarcted areas of patients surviving for 3 to 29 days after stroke (Figure 2Cii and 2Ciii). In the rat, RT-PCR and Western blotting demonstrated no notable changes in INI1 mRNA and protein expression respectively following MCAO. Weak cytoplasmic staining was observed in contralateral neurons but no differences in the level of INI1 neuronal expression occurred following MCAO (data not included). Finally, HFN and HBMEC exposed to OGD and/or reperfusion showed no difference in mRNA and protein levels for INI1 when compared with untreated cells.
                          Table 4

                          Protein expression in infarcted (I) and peri-infarcted (P) areas (Fold increase compared to contralateral hemisphere)

                            

                          PAK1

                          INI1

                          MMP11

                          Patient no.

                          Survival (days)

                          P

                          I

                          P

                          I

                          P

                          I

                          1

                          2

                          2.2

                          1.0

                          1.5

                          1.5

                          1.5

                          1.5

                          2

                          3

                          3.3

                          4.0

                          0.2

                          0.4

                          1.0

                          1.0

                          3

                          3

                          1.0

                          1.0

                          4.2

                          4.3

                          0.7

                          0.7

                          4

                          6

                          1.0

                          1.0

                          4.3

                          5.8

                          1.6

                          1.5

                          5

                          9

                          1.5

                          0.4

                          3.2

                          3.3

                          ND

                          ND

                          6

                          15

                          2.3

                          1.5

                          2.8

                          1.0

                          1.7

                          1.6

                          7

                          15

                          3.0

                          3.2

                          1.7

                          2.0

                          1.0

                          1.0

                          8

                          17

                          1.0

                          1.0

                          1.0

                          1.0

                          1.0

                          1.0

                          9

                          20

                          1.0

                          1.0

                          1.0

                          1.0

                          1.0

                          1.0

                          10

                          26

                          1.5

                          1.5

                          1.0

                          1.6

                          5.1

                          2.2

                          11

                          29

                          1.0

                          1.5

                          2.2

                          2.8

                          1.8

                          3.5

                          12

                          37

                          1.5

                          1.0

                          1.7

                          1.7

                          1.0

                          1.5

                          Total

                          Upregulated

                          7

                          5

                          8

                          8

                          5

                          6

                           

                          Downregulated

                          0

                          1

                          1

                          1

                          1

                          1

                           

                          No change

                          5

                          6

                          3

                          3

                          5

                          4

                           

                          No detection

                          0

                          0

                          0

                          0

                          1

                          1

                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2202-8-93/MediaObjects/12868_2007_Article_391_Fig2_HTML.jpg
                          Figure 2

                          INI1 expression in human brain following stroke. RT-PCR demonstrated an increase in ini1 mRNA levels in infarcted and peri-infarcted areas of pooled samples from patients surviving from 2 to 6 days following stroke (A). Western blotting showed an increase in protein levels in infarcted and peri-infarcted areas of patients surviving for 3 (Bi) and 6 (Bii) days following stroke. Moderate INI1 neuronal staining (arrow) in contralateral areas of a patient surviving for 3 days after stroke (Ci). Strong INI1 staining in cells (arrows) from infarcted areas of a patient surviving for 15 days after stroke (Cii and iii) (C: Contralateral, P: Peri-infarct, I: Infarct).

                          Matrix Metalloproteinase 11 (MMP11)

                          For MMP11, RT-PCR data agreed with the findings from the microarray study, showing increased mRNA levels in infarcted and peri-infarcted tissue from patients surviving 2–20 days following stroke (Figure 3Ai). Western blotting in individual patient samples demonstrated that 6 of 12 patients had elevated MMP11 protein levels (Table 4; Figure 3Bi and 3Bii). The majority of cells from contralateral grey and white matter were not stained for MMP11 (Figure 3Ci). In patients surviving from 3 days to 4 weeks, endothelial cells and neurons from both infarcted and peri-infarcted tissue were stained positive for MMP11 (Figure 3Cii and 3Ciii). In the rat model, RT-PCR confirmed the microarray data for some of the time-points, showing no notable change in mRNA levels at 1 and 12 h but a prolonged upregulation at 3 days following MCAO (Figure 3Aii). Protein levels were elevated at 1 h, 24 h and 3 days, after which they returned to control levels. No staining for MMP11 was seen in contralateral areas (Figure 3Di), but an increase in its expression occurred in neurons following MCAO, in particular at 12 and 24 h (Figure 3Dii). MMP11 mRNA and protein levels remained unchanged in HFN and HBMEC exposed to conditions of oxygen-glucose deprivation.
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2202-8-93/MediaObjects/12868_2007_Article_391_Fig3_HTML.jpg
                          Figure 3

                          MMP11 expression in human and rat brain following stroke. RT-PCR demonstrated an increase in MMP11 mRNA levels in infarcted and peri-infarcted areas of patients surviving from 2 to 6 days following stroke (Ai) and rats at 3 days after MCAO (Aii). Western blotting demonstrated an increase in protein levels in infarcted and peri-infarcted areas of patients surviving for 29 (Bi) and 26 (Bii) days following stroke. Weak MMP11 staining in cells from contralateral areas of a patient surviving for 5 days following stroke (Ci). Blood vessels (Cii) and neurons (Ciii) strongly stained for MMP11 in peri-infarcted areas of a patient surviving for 15 days after stroke (arrows). No MMP11 staining observed in contralateral hemisphere of rat brain at 1 h after MCAO (Di) but neurons from infarcted areas of rat brain were stained positive for MMP11 at 3 days following MCAO (Dii) (C: Contralateral, P: Peri-infarct, I: Infarct).

                          P21-activated kinase 1 (PAK1)

                          RT-PCR confirmed the upregulation of pak1 determined by the microarrays in pooled samples from stroke patients who survived between 2 and 6 days following stroke (Figure 4A). Western blotting showed an upregulation in the protein levels of PAK1 in 6 of 12 patients (Table 4; Figure 4Bi and 4Bii). No staining was seen in contralateral white matter, while, in grey matter, PAK1 stained weakly the cytoplasm of some neurons (Figure 4Di). In patients surviving for 3 days to 4 weeks after stroke, increased PAK1 nuclear staining was seen in neurons in both peri-infarcted and infarcted regions (Figure 4Dii). In the rat, RT-PCR showed no significant changes in the mRNA levels for pak1 at most of the time-points examined. However, Western blotting showed an upregulation in protein levels 1, 12 and 24 h after MCAO, returning to control levels at 3 days, and becoming downregulated at 7 days following MCAO (Figure 4Ci and 4Cii). Weak staining was observed in neurons from the contralateral hemisphere (Figure 4Ei), but an increase in cytoplasmic and nuclear staining in neurons occurred following MCAO, in particular at 1 h and 24 h (Figure 4Eii). Finally, an increase in PAK1 expression was also seen in human foetal neurons following oxygen-glucose deprivation (Figure 4Fi and 4Fii).
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2202-8-93/MediaObjects/12868_2007_Article_391_Fig4_HTML.jpg
                          Figure 4

                          PAK1 expression in human and rat brain following stroke. RT-PCR demonstrated an increase in PAK1 mRNA in infarcted and peri-infarcted areas of pooled samples from patients surviving from 2 to 6 days following stroke (A). Western blotting demonstrated an increase in protein levels in infarcted and peri-infarcted areas of patients surviving for 3 (Bi) and 15 (Bii) days following stroke and in rats at 12 h (Ci) and 24 h (Cii) following MCAO. Weak neuronal (axonal) staining (arrow) observed in contralateral areas of a patient surviving for 15 days following stroke (Di). Strong PAK1 staining in neurons (arrow) and cells with the morphological appearance of glia from infarcted areas of a patient surviving for 3 days following stroke (Dii). No staining observed in contralateral areas of rat brain at 24 h following MCAO (Ei) while strong PAK1 staining was seen in neurons (arrow) and cells with the morphological appearance of glia from infarcted areas of rat brain 1 h following MCAO (Eii). Stronger PAK1 immunofluorescent staining was seen in HFN following OGD (Fii) compared to control (Fi) (C: Contralateral, P: Peri-infarct, I: Infarct).

                          Discussion and Conclusion

                          In the human brain, many differentially expressed genes were observed from 2 to 6 days and from 9 to 20 days after stroke, with the majority being upregulated. The number of deregulated genes declined during 26 to 37 days after stroke, indicating that dynamic changes in gene expression occur during the first days to few weeks in the human postischaemic brain. In the rat brain, few differences were observed at 1 hour, while the number of differentially expressed genes steadily increased with time after MCAO, with a peak after 3 days, supporting the concept of active mechanisms initiated during the acute phase after experimental stroke and lasting for several days. The number of upregulated genes gradually increased, peaking at 3 days, while downregulated genes were detected 24 h after MCAO and increased dramatically until the final measured time-point at 21 days (Figure 1).

                          The limitations of post-mortem brain samples in cDNA microarray analysis concern the small sample size and potential low quality and the genetic heterogeneity and diversity in terms of age, sex and previous medical history within a group of patients [28, 29]. We found that analysis of postischaemic gene expression using a cDNA microarray can allow identification of known and novel transcriptional events, molecular participants and signalling mechanisms in cerebral ischaemia as previously suggested, but can also detect differences in gene expression between distinct organisms.

                          The present gene expression profile study is the first large-scale microarray report showing altered expression of several genes following human stroke. These included genes participating in transcription, apoptosis, inflammation and neuroprotection. Many genes/proteins previously shown to be deregulated following stroke were reported in our study too e.g. IL-10 [30, 31], PDGF [32], STAT3 [33, 34], MAPK1/2 [35]. To test whether our microarray analysis could predict novel candidate genes involved in the cerebral response to ischaemia with possible functional importance and significance in stroke-induced neuronal damage, we measured protein expression and cellular localisation for three induced genes, INI1, PAK1 and MMP11. They were chosen because they showed at least 2-fold mRNA induction and there was no prior published evidence implicating them in human cerebral ischaemia.

                          PAK1 is a downstream Rac effector and a major cyclin-dependent kinase 5 (Cdk5) substrate and target that co-localizes with p35/Cdk5 at neuronal peripheries. P35/Cdk5 causes PAK1 hyperphosphorylation, which results in PAK1 down-regulation and is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons during dendrite development [36]. Based on this evidence, these authors proposed the existence of a neuron-specific signalling complex involving Cdk5/p35-PAK1 that inhibits PAK1 activity in neurons. We have recently provided evidence for a potential role of Cdk5/p35 in the response to ischaemic injury as we showed association of Cdk5 with nuclear damage, by demonstrating co-expression of Cdk5 in TUNEL-positive neurons following human stroke and in propidium iodide-positive human foetal neurons following OGD [37]. Here, we have reported for the first time an upregulation in PAK1 protein levels in human and rat brain samples following MCAO and in HFN following oxygen-glucose deprivation. Although in the animal model PAK1 protein levels returned to normal 3 days following stroke, some patients showed elevated levels for PAK1 at later time-points too. In both human and the animal model, neurons were the predominant type of cells stained positive for PAK1.

                          MMP11 or stromelysin-3 (ST3), first isolated as a breast cancer-associated protease, is not expressed in the majority of normal adult organs but is expressed during a number of pathological processes, including wound healing and atherosclerotic lesions [38, 39]. Although other metalloproteinases have been studied extensively following stroke [40, 41], there is no report of the expression of MMP11 following stroke in vivo or in vitro. Here we report an increase in protein levels of MMP11 following stroke in both human and rat brain, although the increase seen in man remained elevated much longer. Although MMP11 shares many similarities with other MMPs, it also differs in that it exhibits anti-apoptotic properties, a first-known activity for a MMP [42]. Moreover, although it is expressed in many processes involving tissue remodelling, cell migration and cell death, the pathways through which it participates in pathogenesis remain unclear, largely due to the lack of information on its substrates in vivo [43].

                          INI1 is a tumour suppressor gene, thought to exert its tumour suppressor function by mediating cell cycle arrest [44]. It was initially identified as a human homolog of yeast transcriptional activator SNF5 that binds to the HIV-1 integrase and stimulates its DNA-joining activity [45]. Brains of AIDS patients had been shown to manifest neuronal injury and apoptotic-like cell death raising the question about the way HIV-1 resulted in neuronal damage, since neurons themselves are very rarely infected by the virus [46]. Adler et al. [47] also reported an association of the human SNF5/INI1 protein with growth arrest and DNA damage-inducible protein 34 (GADD34) that mediates growth arrest and apoptosis in response to stress signals [48, 49]. Our study is the first to suggest a potential role for INI1 in pathways activated after stroke with a possible role in brain injury. However, in the animal model study, INI1 levels remained unchanged following stroke. The reason for this discrepancy warrants further studies.

                          Many experimental trials of stroke therapies have failed to translate to human clinical trials and one possible way to improve the success rate can be through comparative genomics. As it has been recently commented, it is very surprising that the exciting developments observed in basic and clinical stroke research over the past two decades have occurred in parallel, with too little direct translation between bench and bedside [50]. Here, we have provided substantial evidence that, although the available animal models of MCAO may well be suitable to study the pathophysiological changes following the occlusion of a cerebral vessel, they may not entirely reflect the pathophysiological process through which stroke evolves in humans. The species difference is one of the main reasons accounting for the lack of success of bench to bedside translation in the stroke area. Limitations of our study include the fact that early acute phase changes in gene expression may have been missed since genes induced and returning to normal during the first 48 hours post-ischaemia in man could not have been detected. Moreover, since we analyzed pooled RNA samples, small changes in gene expression occurring in a minority of the samples may have been missed. However, there was only a small overlap of our results with prior studies in experimental stroke involving brain tissue, and the successful identification of novel ischaemia-related genes reported here suggests that performing a further study using whole genome microarrays would be valuable.

                          Methods

                          Human brain autopsy specimens

                          Human brain tissue samples were obtained from 12 patients who died from acute ischaemic stroke, with the approval of the local Ethics Committee and Brain Bank at the Department of Neuropathology, Collegium Medicum, Jagiellonian University, Krakow, Poland. All patients were admitted with large middle cerebral artery strokes confirmed by CT-scan or MRI. The patients, 10 male and 2 female, were aged between 51 and 86 years and had survived between 2–37 days following ischaemic stroke (Table 2). Routine blood parameters were determined on admission. Full clinical examinations, including NIH Stroke Scale, were also carried out on admission. Excluded from the study were patients with recent history of head trauma, major cardiac, renal, hepatic or cancerous disease and obvious signs of infection after admission. Immediately after death the body was put in a cold chamber and tissue was collected within 6 h of death. Tissue samples were taken from infarct and peri-infarcted zones while controls were obtained from the contralateral hemisphere at the same time. The peri-infarcted areas were defined in tissue sections as the tissue immediately surrounding the infarcted core which contained some necrotic cells and showed evidence of tissue disorganisation confirmed by histology. Sections were stained with 2,3,5-triphenyltetrazolium chloride which stains active mitochondria pink; therefore, non stained areas represented stroke affected cortical regions (data not included). Tissue specimens were immediately frozen in liquid nitrogen, kept at -70°C and a portion of each sample was processed for histology and stained with haematoxylin and eosin to determine tissue morphology [51].

                          Rat middle cerebral artery occlusion

                          Stroke experiments were performed on female Sprague-Dawley rats (weight: 230–270 g) as they suffer less than male during ischaemia. Cerebral ischaemia was produced using a modified method of Baron [52] by distal, permanent occlusion of the MCA by electrocautery as described elsewhere [53, 54]. The mortality in this model is very low. Sets of six animals (3 for morphological studies and 3 pooled together) for each time-point were sacrificed at 1 h, 4 h, 12 h, 24 h and 3, 7 and 21 days.

                          In vitro oxygen-glucose deprivation (OGD)

                          Human brain microvascular endothelial cells (HBMEC) were obtained from TCS CellWorks (Buckingham, UK) and cultured according to the supplier's instructions. Human foetal (cerebral cortical) neurons (HFN) were extracted and cultured with permission from the Local Ethics Committee. Brain tissue from foetus specimens of 14–19 weeks gestational age, legally aborted and with the appropriate written consent, were collected in cold preservation medium and cells were isolated and cultured as described elsewhere [55]. For OGD experiments, the culture medium was replaced by glucose-free medium containing 2% foetal bovine serum (TCS CellWorks, Buckingham, UK) and cells were cultured at 37°C in a humidified chamber with 94% N2, 1% O2, and 5% CO2 for 6 h (HBMEC) or 95% N2 and 5% CO2 for 14 h (HFN) followed by 24 h reperfusion in fresh medium containing 4.5 g/l glucose. This resulted in approximately 30% of cells undergoing apoptosis after OGD and 60% following reoxygenation, as determined from our pilot studies. Cells cultured in normoxic conditions without glucose deprivation were used as controls. In some experiments, propidium iodide (10 μg/ml) was added to the cultures 1 h before the end of the experiment to stain dead and dying cells.

                          cDNA microarrays

                          We established mRNA expression profiles of the damaged brain tissues between 2 to 6 days, 9 to 20 days, and 26 to 37 days after stroke in human patients and 1, 4, 12, 24 hours and 3, 7 and 21 days after the ischaemic insult in rats. The corresponding samples from the non-ischemic control hemisphere were used to measure the normal mRNA abundance of the modulated genes in each tissue at each time point. RNA from three stroke patients was pooled for each patient survival group while RNA from three MCAO rats was also pooled at each time-point to improve yields in preparation of poly A+ RNA. Although pooling was previously thought to affect data quality, Kendziorski et al. [56] have recently shown that inference was not adversely affected by pooling. The different patient groups were selected to match the three physiological stages following stroke i.e. the inflammatory (lasting up to a maximum of 5–6 days), the proliferative (lasting up to three weeks following stroke) and the remodelling/maturation (starting during the third or fourth week).

                          RNA was extracted according to the manufacturer's protocols (BD Biosciences, Oxfordshire, UK) and its quality was measured spectrophotometically. The protocol recommended by Clontech in their Atlas 1.2 microarray kit was used without any modification. Briefly, RNA was reverse-transcribed to cDNA, 32P-labelled and applied to the array for overnight hybridisation at 68°C. Following washing, the array was exposed to a phosphorimaging plate for 12–72 hours and data analysis was performed using the AtlasImage 1.5 software. The results were normalized using two housekeeping genes, ubiquitin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As in the majority of microarray studies mentioned before, only those genes upregulated > 2-fold or downregulated < 0.5-fold were counted as deregulated and taken into consideration. The microarray data are available in Gene Expression Omnibus under the accession number GSE9391.

                          Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

                          Gene expression was examined by semi-quantitative RT-PCR with standard reaction conditions of a 10 min denaturation at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at primer-specific annealing temperatures (Table 5) and 1 min at 72°C and a final 10 min extension step at 72°C. Samples without cDNA were used as negative controls and the products were visualized by agarose gel electrophoresis (1.5% w/v) and DNA stained with ethidium bromide (10 mg/ml). All experiments were carried out at 25, 30, 35 and 40 cycles to ensure the semi-quantitative nature of the results. The results were normalized using housekeeping gene GAPDH and semi-quantitavely analyzed using Scion Imaging Software version 4.02 (Scion Corporation, Maryland, USA). Sense and antisense oligonucleotide primers containing 18–27 nucleotides based on previously reported mRNA sequences in the GenBank depository were designed with the aid of the Primer3 Output Program (Version 0.2). InVitrogen plc. (Paisley, UK) synthesized the primer sets (Table 5).
                          Table 5

                          Primer sequences

                          Gene

                          Species

                          Primer Sequence

                          T annealing

                          mmp11

                          Human

                          5'-TAAAGGTATGGAGCGATGTGAC-3' (forward)

                          58°C

                          mmp11

                           

                          5'-TGGGTAGCGAAAGGTGTAGAAG-3' (reverse)

                           

                          mmp11

                          Rat

                          5'-GATGGAGGCCAGCTAGTCAG-3' (forward)

                          60°C

                          mmp11

                           

                          5'-ATGGTACATGACCACGCAGA-3' (reverse)

                           

                          ini1

                          Human

                          5'-ACCCTGTCCAACAGCTCCCA-3' (forward)

                          64°C

                          ini1

                           

                          5'-GGCCCAATCTTCTGAGATGC-3' (reverse)

                           

                          ini1

                          Rat

                          5'-CCTGGGGCTCCTATACAAAA-3' (forward)

                          60°C

                          ini1

                           

                          5'-CCATGACCGAGCAAATGAC-3' (reverse)

                           

                          pak1

                          Human

                          5'-GCTGTTCTGGATGTGTTGGA-3' (forward)

                          60°C

                          pak1

                           

                          5'-TCTGCTCTGGGGTTATCTGTG-3' (reverse)

                           

                          pak1

                          Rat

                          5'-AGCAAAAGAGGCAACCAAGA-3' (forward)

                          60°C

                          pak1

                           

                          5'-GGGTAAGGAATGGGATGGTT-3' (reverse)

                           

                          gapdh

                          Human

                          5'-ATGATCTTGAGGCTGTTG-3' (forward)

                          58°C

                          gapdh

                           

                          5'-CTCAGACACCATGGGGAA-3' (reverse)

                           

                          Protein extraction and Western blotting

                          Proteins were extracted from tissues and the protein concentration of each sample was determined using the BioRad assay. For Western blotting, 10 μg of protein were separated by SDS-PAGE (13% w/v) and the proteins were electro-blotted onto nitrocellulose filters as described previously [57]. Filters were blocked in 1% w/v bovine serum albumin (BSA) in Tris-buffered saline Tween (TBS Tween) and stained overnight at 4°C with antibodies to the following proteins (obtained from Autogen Bioclear, Wiltshire, UK, unless stated otherwise) diluted in 1% BSA: MMP11 (CalBiochem; 1:500), PAK1 (1:500), INI1 (1:500), and α-actin (Sigma, 1:1000) used as a loading control. Membranes were washed in TBS-Tween before staining with the appropriate peroxidase-conjugated secondary antibody, diluted 1:1000 in 5% w/v milk in TBS-Tween for l h. Blots were developed with the ECL detection system (Amersham, UK). The relative intensities of the bands were measured in an LKB densitometer. Results are semi-quantitative and are given as a numerical (fold) change compared to the control (contralateral tissue) which was given an arbitrary value of 1.0. All experiments were performed twice and a representative example of patient(s) showing an increase in protein expression is given.

                          Immunohistochemistry/Immunofluorescence

                          Paraffin-embedded tissue samples were processed and serial 5 μm sections were cut. The Avidin-Biotin Peroxidase (ABC Vectastain kit, Vector Laboratories, Peterborough, UK) method was used and antibodies to MMP11, PAK1 and INI1 were used at a dilution of 1:50. Paraffin-embedded sections were deparaffinized, rehydrated and boiled for 10 min in an antigen unmasking solution of concentrated citric acid pH 6.0 as described elsewhere [57]. Slides were incubated in 0.5% v/v H2O2 in methanol for 30 min, with normal serum for 20 min and then with a primary antibody (diluted in normal serum) for 30 min, followed by 30-min incubation with biotinylated secondary antibody (diluted 1:50) and finally with ABC complex (diluted 1:50) for 30 min at RT. Staining was completed after incubation with DAB substrate chromogen solution for 3–10 min. Slides were counterstained with haematoxylin, dehydrated, cleared and mounted in DPX. For immunofluorescence, cultured cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton ×100 for 10 min, blocked with normal serum and stained with the primary antibody as above, followed by 1 h incubation with Alexa-fluor conjugated dye at RT. Negative control slides were performed in parallel, where primary antibody was replaced with washing buffer and processed as above (data not included).

                          Abbreviations

                          GAPDH: 

                          glyceraldehyde 3-phosphate dehydrogenase

                          HBMEC: 

                          human brain microvascular endothelial cells

                          HFN: 

                          human foetal neurons

                          INI1: 

                          integrase interactor 1

                          MCAO: 

                          middle cerebral artery occlusion

                          MMP11: 

                          matrix metalloproteinase

                          OGD: 

                          oxygen-glucose deprivation

                          PAK1: 

                          p21-activated kinase 1.

                          Declarations

                          Acknowledgements

                          This work was supported by the Higher Education Funding Council for England (HEFCE) and the Research Institute for Health and Social Change (RIHSC). We also thank Mick Hoult for his assistance in the preparation of this manuscript.

                          Authors’ Affiliations

                          (1)
                          School of Biology, Chemistry and Health Science, John Dalton Building, Manchester Metropolitan University
                          (2)
                          Department of Neurology, Stroke Unit, Hospital Universitari de Bellvitge (HUB), Fundacio IDIBELL, L' Hospitalet de Llobregat
                          (3)
                          Departamento de Farmacologia i Toxicologia Institut d' Investigacions Biomediques de Barcelona (IIBB), CSIC-IDIBAPS
                          (4)
                          Department of Pathology, Medical School, University of Manchester and Christie Hospital

                          References

                          1. Mitsios N, Gaffney J, Kumar P, Krupinski J, Kumar S, Slevin M: Pathophysiology of acute ischaemic stroke: An analysis of common signalling mechanisms and identification of new molecular targets. Pathobiology 2006,73(4):159–175.View ArticlePubMed
                          2. Slevin M, Krupinski J, Kumar P, Gaffney J, Kumar S: Gene activation and protein expression following ischaemic stroke: strategies towards neuroprotection. Journal Of Cellular And Molecular Medicine 2005,9(1):85–102.View ArticlePubMed
                          3. Schaller B, Graf R: Cerebral ischemia and reperfusion: The pathophysiologic concept as a basis for clinical therapy. Journal Of Cerebral Blood Flow And Metabolism 2004,24(4):351–371.PubMed
                          4. Slevin M, Kumar P, Gaffney J, Kumar S, Krupinski J: Can angiogenesis be exploited to improve stroke outcome? Mechanisms and therapeutic potential. Clinical Science 2006,111(3):171–183.View ArticlePubMed
                          5. Longa EZ, Weinstein PR, Carlson S, Cummins R: Reversible Middle Cerebral-Artery Occlusion Without Craniectomy In Rats. Stroke 1989,20(1):84–91.PubMed
                          6. Hasselblatt M, Jeibmann A, Riesmeier B, Maintz D, Schabitz WR: Granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor expression in human ischemic stroke. Acta Neuropathologica 2007,113(1):45–51.View ArticlePubMed
                          7. Rosell A, Ortega-Aznar A, Alvarez-Sabin J, Fernandez-Cadenas I, Ribo M, Molina CA, Lo EH, Montaner J: Increased brain expression of matrix metalloproteinase-9 after ischemic and hemorrhagic human stroke. Stroke 2006,37(6):1399–1406.View ArticlePubMed
                          8. Kim YD, Sohn NW, Kang CH, Soh Y: DNA array reveals altered gene expression in response to focal cerebral ischemia. Brain Research Bulletin 2002,58(5):491–498.View ArticlePubMed
                          9. Rao VLR, Bowen KK, Dhodda VK, Song GQ, Franklin JL, Gavva NR, Dempsey RJ: Gene expression analysis of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia. Journal Of Neurochemistry 2002,83(5):1072–1086.View Article
                          10. Roth A, Gill R, Certa U: Temporal and spatial gene expression patterns after experimental stroke in a rat model and characterization of PC4, a potential regulator of transcription. Molecular And Cellular Neuroscience 2003,22(3):353–364.View ArticlePubMed
                          11. Lippoldt A, Reichel A, Moenning U: Progress in the identification of stroke-related genes - Emerging new possibilities to develop concepts in stroke therapy. Cns Drugs 2005,19(10):821–832.View ArticlePubMed
                          12. Ford G, Xu ZF, Gates A, Jiang J, Ford BD: Expression Analysis Systematic Explorer (EASE) analysis reveals differential gene expression in permanent and transient focal stroke rat models. Brain Research 2006,1071(1):226–236.View ArticlePubMed
                          13. Jin KL, Mao XO, Eshoo MW, Nagayama T, Minami M, Simon RP, Greenberg DA: Microarray analysis of hippocampal gene expression in global cerebral ischemia. Annals Of Neurology 2001,50(1):93–103.View ArticlePubMed
                          14. Kim JB, Piao CS, Lee KW, Han PL, Ahn JI, Lee YS, Lee JK: Delayed genomic responses to transient middle cerebral artery occlusion in the rat. Journal Of Neurochemistry 2004,89(5):1271–1282.View ArticlePubMed
                          15. Lu XCM, Williams AJ, Yao C, Berti R, Hartings JA, Whipple R, Vahey MT, Polavarapu RG, Woller KL, Tortella FC, Dave JR: Microarray analysis of acute and delayed gene expression profile in rats after focal ischemic brain injury and reperfusion. Journal Of Neuroscience Research 2004,77(6):843–857.View ArticlePubMed
                          16. Schmidt-Kastner R, Zhang BT, Belayev L, Khoutorova L, Amin R, Busto R, Ginsberg MD: DNA microarray analysis of cortical gene expression during early recirculation after focal brain ischemia in rat. Molecular Brain Research 2002,108(1–2):81–93.View ArticlePubMed
                          17. Soriano MA, Tessier M, Certa U, Gill R: Parallel gene expression monitoring using oligonucleotide probe arrays of multiple transcripts with an animal model of focal ischemia. Journal Of Cerebral Blood Flow And Metabolism 2000,20(7):1045–1055.PubMed
                          18. Tang Y, Lu AG, Aronow BJ, Wagner KR, Sharp FR: Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia. European Journal Of Neuroscience 2002,15(12):1937–1952.View ArticlePubMed
                          19. Moore DF, Li H, Jeffries N, Wright V, Cooper RA, Elkahloun A, Gelderman MP, Zudaire E, Blevins G, Yu H, Goldin E, Baird AE: Using peripheral blood mononuclear cells to determine a gene expression profile of acute ischemic stroke - A pilot investigation. Circulation 2005,111(2):212–221.View ArticlePubMed
                          20. Tang Y, Xu HC, Du XL, Lit L, Walker W, Lu AG, Ran RQ, Gregg JP, Reilly M, Pancioli A, Khoury JC, Sauerbeck LR, Carrozzella JA, Spilker J, Clark J, Wagner KR, Jauch EC, Chang DJ, Verro P, Broderick JP, Sharp FR: Gene expression in blood changes rapidly in neutrophils and monocytes after ischemic stroke in humans: a microarray study. Journal Of Cerebral Blood Flow And Metabolism 2006,26(8):1089–1102.View ArticlePubMed
                          21. Hynd MR, Lewohl JM, Scott HL, Dodd PR: Biochemical and molecular studies using human autopsy brain tissue. Journal Of Neurochemistry 2003,85(3):543–562.View ArticlePubMed
                          22. Schramm M, Falkai P, Tepest R, Schneider-Axmann T, Przkora R, Waha A, Pietsch T, Bonte W, Bayer TA: Stability of RNA transcripts in post-mortem psychiatric brains. Journal Of Neural Transmission 1999,106(3–4):329–335.View ArticlePubMed
                          23. Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ: Apoptosis dominant in the periinfarct area of human ischaemic stroke - a possible target of antiapoptotic treatments. Brain 2006, 129:189–199.View ArticlePubMed
                          24. Sairanen T, Ristimaki A, Karjalainen-Lindsberg ML, Paetau A, Kaste M, Lindsberg PJ: Cyclooxygenase-2 is induced globally in infarcted human brain. Annals Of Neurology 1998,43(6):738–747.View ArticlePubMed
                          25. Castensson A, Emilsson L, Preece P, Jazin E: High-resolution quantification of specific mRNA levels in human brain autopsies and biopsies. Genome Research 2000,10(8):1219–1229.View ArticlePubMed
                          26. Almeida A, Thiery JP, Magdelenat H, Radvanyi F: Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling. Analytical Biochemistry 2004,328(2):101–108.View ArticlePubMed
                          27. Vikman P, Edvinsson L: Gene expression profiling in the human middle cerebral artery after cerebral ischemia. European Journal Of Neurology 2006,13(12):1324–1332.View ArticlePubMed
                          28. Iwamoto K, Kato T: Gene expression profiling in schizophrenia and related mental disorders. Neuroscientist 2006,12(4):349–361.View ArticlePubMed
                          29. Mirnics K, Pevsner J: Progress in the use of microarray technology to study the neurobiology of disease. Nature Neuroscience 2004,7(5):434–439.View ArticlePubMed
                          30. Perini F, Morra M, Alecci M, Galloni E, Marchi M, Toso V: Temporal profile of serum anti-inflammatory and pro-inflammatory interleukins in acute ischemic stroke patients. Neurological Sciences 2001,22(4):289–296.View ArticlePubMed
                          31. Tarkowski E, Rosengren L, Blomstrand C, Wikkelso C, Jensen C, Ekholm S, Tarkowski A: Intrathecal release of pro- and anti-inflammatory cytokines during stroke. Clinical And Experimental Immunology 1997,110(3):492–499.View ArticlePubMed
                          32. Krupinski J, Issa R, Bujny T, Slevin M, Kumar P, Kumar S, Kaluza J: A putative role for platelet-derived growth factor in angiogenesis and neuroprotection after ischemic stroke in humans. Stroke 1997,28(3):564–573.PubMed
                          33. Choi JS, Kim SY, Cha JH, Choi YS, Sung KW, Oh ST, Kim ON, Chung JW, Chun MH, Lee SB, Lee MY: Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia. Glia 2003,41(3):237–246.View ArticlePubMed
                          34. Justicia C, Gabriel C, Planas AM: Activation of the JAK/STAT pathway following transient focal cerebral ischemia: Signaling through Jak1 and Stat3 in astrocytes. Glia 2000,30(3):253–270.View ArticlePubMed
                          35. Slevin M, Krupinski J, Slowik A, Rubio F, Szczudlik A, Gaffney J: Activation of MAP kinase (ERK-1/ERK-2), tyrosine kinase and VEGF in the human brain following acute ischaemic stroke. Neuroreport 2000,11(12):2759–2764.View ArticlePubMed
                          36. Nikolic M, Chou MM, Lu WG, Mayer BJ, Tsai LH: The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity. Nature 1998,395(6698):194–198.View ArticlePubMed
                          37. Mitsios N, Pennucci R, Krupinski J, Sanfeliu C, Gaffney J, Kumar P, Kumar S, Juan-Babot O, Slevin M: Expression of cyclin-dependent kinase 5 mRNA and protein in the human brain following acute ischemic stroke. Brain Pathology 2007,17(1):11–23.View ArticlePubMed
                          38. Schonbeck U, Mach F, Sukhova GK, Atkinson E, Levesque E, Herman M, Graber P, Basset P, Libby P: Expression of stromelysin-3 in atherosclerotic lesions: Regulation via CD40-CD40 ligand signaling in vitro and in vivo. Journal Of Experimental Medicine 1999,189(5):843–853.View ArticlePubMed
                          39. Wolf C, Chenard MP, Degrossouvre PD, Bellocq JP, Chambon P, Basset P: Breast-Cancer Associated Stromelysin-3 Gene Is Expressed In Basal-Cell Carcinoma And During Cutaneous Wound-Healing. Journal Of Investigative Dermatology 1992,99(6):870–872.View ArticlePubMed
                          40. Montaner J, Alvarez-Sabin J, Molina C, Angles A, Abilleira S, Arenillas J, Gonzalez MA, Monasterio J: Matrix metalloproteinase expression after human cardioembolic stroke - Temporal profile and relation to neurological impairment. Stroke 2001,32(8):1759–1766.PubMed
                          41. Montaner J, Alvarez-Sabin J, Molina CA, Angles A, Abilleira S, Arenillas J, Monasterio J: Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke. Stroke 2001,32(12):2762–2767.View ArticlePubMed
                          42. Matziari M, Dive V, Yiotakis A: Matrix metalloproteinase 11 (MMP-11; stromelysin-3) and synthetic inhibitors. Medicinal Research Reviews 2007., 10.1002/med.20066:
                          43. Wei L, Shi YB: Matrix metalloproteinase stromelysin-3 in development and pathogenesis. Histology And Histopathology 2005,20(1):177–185.PubMed
                          44. Zhang ZK, Davies KP, Allen J, Zhu L, Pestell RG, Zagzag D, Kalpana GV: Cell cycle arrest and repression of cyclin D1 transcription by INI1/hSNF5. Molecular And Cellular Biology 2002,22(16):5975–5988.View ArticlePubMed
                          45. Kalpana GV, Marmon S, Wang WD, Crabtree GR, Goff SP: Binding And Stimulation Of Hiv-1 Integrase By A Human Homolog Of Yeast Transcription Factor Snf5. Science 1994,266(5193):2002–2006.View ArticlePubMed
                          46. Lipton SA: Neuronal injury associated with HIV-1: Approaches to treatment. Annual Review Of Pharmacology And Toxicology 1998, 38:159–177.View ArticlePubMed
                          47. Adler HT, Chinery R, Wu DY, Kussick SJ, Payne JM, Fornace AJ, Tkachuk DC: Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins. Molecular And Cellular Biology 1999,19(10):7050–7060.PubMed
                          48. Hollander MC, Sheikh MS, Yu K, Zhan QM, Iglesias M, Woodworth C, Fornace AJ: Activation of Gadd34 by diverse apoptotic signals and suppression of its growth inhibitory effects by apoptotic inhibitors. International Journal Of Cancer 2001,96(1):22–31.View Article
                          49. Hollander MC, Zhan QM, Bae I, Fornace AJ: Mammalian GADD34, an apoptosis- and DNA damage-inducible gene. Journal Of Biological Chemistry 1997,272(21):13731–13737.View ArticlePubMed
                          50. Dirnagl U: Bench to bedside: the quest for quality in experimental stroke research. Journal Of Cerebral Blood Flow And Metabolism 2006,26(12):1465–1478.View ArticlePubMed
                          51. Eke A, Conger KA, Anderson M, Garcia JH: Histologic Assessment Of Neurons In Rat Models Of Cerebral-Ischemia. Stroke 1990,21(2):299–304.PubMed
                          52. Baron JC: Perfusion thresholds in human cerebral ischemia: Historical perspective and therapeutic implications. Cerebrovascular Diseases 2001, 11:2–8.View ArticlePubMed
                          53. Krupinski J, Lopez E, Marti E, Ferrer I: Expression of caspases and their substrates in the rat model of focal cerebral ischemia. Neurobiology Of Disease 2000,7(4):332–342.View ArticlePubMed
                          54. Krupinski J, Slevin M, Marti E, Catena E, Rubio F, Gaffney J: Time-course phosphorylation of the mitogen activated protein (MAP) kinase group of signalling proteins and related molecules following middle cerebral artery occlusion (MCAO) in rats. Neuropathology And Applied Neurobiology 2003,29(2):144–158.View ArticlePubMed
                          55. Cristofol RM, Gasso S, Vilchez D, Pertusa M, Rodriguez-Farre E, Sanfeliu C: Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: A comparative study with rat neuronal cultures and human cell lines. Toxicology Letters 2004,152(1):35–46.View ArticlePubMed
                          56. Kendziorski C, Irizarry RA, Chen KS, Haag JD, Gould MN: On the utility of pooling biological samples in microarray experiments. Proceedings Of The National Academy Of Sciences Of The United States Of America 2005,102(12):4252–4257.View ArticlePubMed
                          57. Mitsios N, Gaffney J, Krupinski J, Mathias R, Wang QY, Hayward S, Rubio F, Kumar P, Kumar S, Slevin M: Expression of signaling molecules associated with apoptosis in human ischemic stroke tissue. Cell Biochemistry And Biophysics 2007,47(1):73–85.PubMed

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                          This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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