Introduction
P-glycoprotein(P-gp) is expressed not only in tumor cells but also in some normal tissues including brain capillary endothelium. The mouse mdr1a gene encoding P-gp is predominantly expressed in the brain. P-gp can protect the organs against toxic xenobiotic compounds which are excreted into urine, bile, and the intestinal lumen, and also can prevent their accumulations in critical organs such as brain and testis. However, there are few studies to search for the function of P-gp related to brain ischemia. We investigated the functional effects of P-gp on focal cerebral ischemia using mdr1a knockout (ko) mice.
Methods
Mdr1a ko mice and FVB wild mice were used. We prepared the middle cerebral artery occlusion model for 30 min by using a 6-0 siliconized filament in all mice. The anesthesia was maintained with isoflurane in N2O and O2 by an inhalation mask during the surgical procedure. In order to monitor regional cerebral blood flow (rCBF) continuously, a laser-Doppler flowmetry probe was fixed to the intact skull. Forty eight hours after reperfusion, all mice were deeply anesthetized with isoflurane. Then thoractomies were performed to expose the heart. The brain was fixed with 4% paraformaldehyde through the heart. After that, 40 μ coronal sections were made from the brain. Those sections were stained with monoclonal mouse antibody against mouse neuronal nuclei (NeuN) for identification of the infarct area. For measuring the infarcted volumes, 5 sections at approximately 0.7 mm distance chosen, immunostained with antibody against NeuN, scanned and infracted areas were measured using NIH image version 1.55. Infarcted volumes were calculated using by the three-dimensional reconstruction. The direct arterial blood pressure was measured using a cannula inserted into the tail artery connected with a transducer and micropump in some mdr1a ko mice and FVB wild mice. Heart rate was calculated from arterial pressure pulses. Arterial blood samples were obtained from the same line and analyzed for the partial PaO2, PaCO2, Base Excess and hemoglobin before ischemia and 30 min after reperfusion. In order to evaluate differences in cerebrovascular anatomy between mdr1a ko mice and FVB wild mice, thoracotomy was performed on anesthetized animals and India ink was injected.
Results
The mean infarction volumes seen in mdr1a ko mice and FVB wild mice were 25% and 40%, respectively. Infarction volume of mdr1a ko mice group was significantly smaller than that seen in FVB wild mice group. rCBF, blood pressure and heart rate did not differ between groups. There are no significant differences in PaO2, PaCO2, Base Excess and hemoglobin before ischemia and 30 min after reperfusion. India ink staining of cerebrovascular anatomy of the circle of Willis demonstrated that there were no gross anatomic differences in the vascular pattern of the cerebral circulation, with intact posterior communicating arteries in both groups.
Conclusion
This study demonstrated that the neuronal damage of mutant mice without P-gp encoded by mdr1a was significantly smaller than that of wild mice. These results indicate that P-gp leads to exacerbate cerebral infarction in brain ischemia.