Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs

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 beta-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.