Background
Cerebral edema contributes significantly to morbidity and death associated with brain ischemia. Recently, aquaporin 1, 4 and 9 have been identified as the three main water channels in the rodent brain (Badaut et al., 2002, J Cereb Blood Flow Metab; 22(4):367). Different studies show that AQP4 and 9 could be implicated in water movements during the formation and resolution of the brain edema after stroke. The role of each AQP in the water movement in the edema formation and resolution is still unclear and debated. To clarify the role of the AQPs in the edema formation after ischemia, we have compared the time course of the expression of AQP1, 4, and 9 with the edema formation after a transient focal brain ischemia in mouse.
Methods
This investigation was realised by immunocytochemistry on mouse brain slices after focal transient ischemia (30 min) induced by occlusion of the middle cerebral artery by a silicon coated filament. Brain sections were stained by antibodies against AQP1, 4, 9 (Chemicon), GFAP (sigma) and MAP2 (sigma). The staining was done at 1 h, 6 h, 24 h, 48 h and 7days after the occlusion. The edema was evaluated by measuring the swelling of the ischemic hemisphere on haematoxylin-eosin stained slices. The levels of expression of AQPs were quantified in the core of ischemia and in the border of the lesion (“penumbra”) delimited on MAP2 stained slices.
Results
The brain swelling was maximal at 1hour (hemispheric volume increased by 8. 4%±3) and at 48hours (by 10.1%±2.5) after the ischemia. The size of the ischemic hemisphere was decreased (−3.5%±1) 7 days after the occlusion. AQP1, mainly expressed on the choroid plexus, did not show a modification of expression after ischemia. AQP4 expression, present on astrocytic endfeet, was increased 1 h (117%± 20, n=3, P<0.05, T-test) after occlusion and returned to the basal level at 24 h (95%±16, n=3) in the core and penumbra. In the penumbra, 48 h after the occlusion, the AQP4 expression was increased on the whole cell, without the normal polarization, (112%±10, n=3, P<0.05, T-test). After 7days, the AQP4 level was the lowest 91%±10. AQP9, supposedly implicated in the glycerol and lactate diffusion, showed a significant induction with 24±6 positive astrocytes in the penumbra versus 5± 1 in the contralateral hemisphere. This expression was increased with time and there were 69± 24 positive astrocytes at 7 days (versus, 3±2 in the contralateral hemisphere). Neuronal AQP9 expression was not modified.
Conclusions
The variations of the level of AQP4 were correlated in time with the variations in brain swelling, in contrast with the AQP9 expression that increased gradually with time. AQP9 was highly expressed on all reactive astrocytes 7 days after ischemia. This study suggests that AQP4 could play a major role in the edema formation and resolution in contrast with AQP1 and 9. AQP9, as suggested by our previous studies, could be implicated in the elimination of the excess of lactate or glycerol.