The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR.
BACKGROUND
ADAMTS1 and ADAMTS8 are proteases involved in extracellular matrix proteolysis and antiangiogenesis, but little is known about their expression and function in cerebral ischemia. We investigated the changes in ADAMTS1 and ADAMTS8 in a rat model of permanent middle cerebral artery occlusion (pMCAO). The expressions of glyseraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, cyclophilin, and RPL13A were examined in order to validate the appropriate housekeeping genes for a long duration after inducing cerebral ischemia.


METHODS
Male Sprague-Dawley rats were subjected to pMCAO, and ischemic penumbra was collected at 2, 24 h, 3, 7, and 21 days after inducing ischemia, ADAMTS1, ADAMTS8, and the four housekeeping genes were quantified using real-time polymerase chain reaction (PCR).


RESULTS
The expression of beta-actin increased up to 21 days, and that of GAPDH decreased at 24 h after pMCAO, with no statistically significant changes in RPL13A and cyclophilin being detected. ADAMTS1 mRNA increased at 2 h after pMCAO, peaked at 24 h, and remained at a high level until 21 days. The expression of ADAMTS8 mRNA decreased at 2 and 24 h after pMCAO, followed by a slight increase at 3 days, and then decreased again at 7 days.


CONCLUSIONS
The results suggest that RPL13A and cyclophilin are two appropriate housekeeping genes for the rat pMCAO model up to 21 days. ADAMTS1 mRNA levels increased, but ADAMTS8 decreased after pMCAO. Our data provide new insight into the mechanism of brain ischemia and self-repair after injury.
Tian
Y F
YF
Institute of Neurobiology, Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi, China.
Zhang
P B
PB
Xiao
X L
XL
Zhang
J S
JS
Zhao
J J
JJ
Kang
Q Y
QY
Chen
X L
XL
Qiu
F
F
Liu
Y
Y
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071096:ADAMTS Proteins; D000071097:ADAMTS1 Protein; D000818:Animals; D002545:Brain Ischemia / Q000201:enzymology*; D004195:Disease Models, Animal; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D020768:Middle Cerebral Artery; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D012333:RNA, Messenger; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; D000071097:ADAMTS1 Protein; C571366:ADAMTS8 protein, rat; C486324:Adamts1 protein, rat; C498472:Adamts8 protein, mouse