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

Mitsios N, Saka M, Krupinski J, Pennucci R, Sanfeliu C, Wang Q, Rubio F, Gaffney J, Kumar P, Kumar S, Sullivan M, Slevin M - BMC Neurosci (2007)

Bottom Line: Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases.Interestingly, the mean fold changes were much higher in the human.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biology, Chemistry and Health Science, John Dalton Building, Manchester Metropolitan University, Chester Street, Manchester, UK. n.mitsios@mmu.ac.uk

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.

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Related in: MedlinePlus

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).
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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).

Mentions: 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).


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

Mitsios N, Saka M, Krupinski J, Pennucci R, Sanfeliu C, Wang Q, Rubio F, Gaffney J, Kumar P, Kumar S, Sullivan M, Slevin M - BMC Neurosci (2007)

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).
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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).
Mentions: 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).

Bottom Line: Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases.Interestingly, the mean fold changes were much higher in the human.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biology, Chemistry and Health Science, John Dalton Building, Manchester Metropolitan University, Chester Street, Manchester, UK. n.mitsios@mmu.ac.uk

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.

Show MeSH
Related in: MedlinePlus