TY - JOUR
T1 - Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs
AU - Aihara, Yasuo
AU - Jahromi, Babak S.
AU - Yassari, Reza
AU - Takahashi, Masataka
AU - Macdonald, R. Loch
N1 - Funding Information:
This work was supported by grants (to R.L.M.) from the American Heart Association, the National Institutes of Health (NS25946) and the Brain Research Foundation. B.S.J. was supported by the Canadian Institutes of Health Research and the American Association of Neurological Surgeons.
PY - 2008/7/15
Y1 - 2008/7/15
N2 - Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after subarachnoid hemorrhage (SAH) had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross-sections of basilar arteries were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) and 4′,6-diamidino-2-phenylindole (DAPI). Positively stained cells were counted and numbers obtained were normalized to the cross-sectional area. The results were compared to normal dog basilar arteries contracted pharmacologically in vitro. SAH resulted in significant vasospasm (P < 0.001 for each, paired t-tests). There were significant increases in mRNA for β-actin (441%, P = 0.01), glyceraldehyde-3-phosphate dehydrogenase (566%, P = 0.007) and 18S ribosomal RNA (320%, P = 0.025) 7 days after SAH. Total mRNA was increased 7 days after SAH relative to genomic DNA (157%, P = 0.009). There were significant increases in the number of cells in the tunica media and adventitia of arteries after SAH and a significant decrease in the media after contraction in vitro. Cells in the tunica media and adventitia labeled with PCNA were significantly increased at both times after SAH. Transcripts for housekeeping genes are increased after SAH, making standardization to them potentially invalid. The increase is due to proliferation of cells in the adventitia and increased total mRNA in the media and adventitia.
AB - Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after subarachnoid hemorrhage (SAH) had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross-sections of basilar arteries were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) and 4′,6-diamidino-2-phenylindole (DAPI). Positively stained cells were counted and numbers obtained were normalized to the cross-sectional area. The results were compared to normal dog basilar arteries contracted pharmacologically in vitro. SAH resulted in significant vasospasm (P < 0.001 for each, paired t-tests). There were significant increases in mRNA for β-actin (441%, P = 0.01), glyceraldehyde-3-phosphate dehydrogenase (566%, P = 0.007) and 18S ribosomal RNA (320%, P = 0.025) 7 days after SAH. Total mRNA was increased 7 days after SAH relative to genomic DNA (157%, P = 0.009). There were significant increases in the number of cells in the tunica media and adventitia of arteries after SAH and a significant decrease in the media after contraction in vitro. Cells in the tunica media and adventitia labeled with PCNA were significantly increased at both times after SAH. Transcripts for housekeeping genes are increased after SAH, making standardization to them potentially invalid. The increase is due to proliferation of cells in the adventitia and increased total mRNA in the media and adventitia.
KW - Cerebral vasospasm
KW - Gene expression
KW - Housekeeping genes
KW - Subarachnoid hemorrhage
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U2 - 10.1016/j.jneumeth.2008.03.020
DO - 10.1016/j.jneumeth.2008.03.020
M3 - Article
C2 - 18490059
AN - SCOPUS:44649127349
SN - 0165-0270
VL - 172
SP - 1
EP - 7
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 1
ER -