Our previous study demonstrated that pharmacological inhibition of the
Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) during
ischemia and reperfusion attenuated neuronal damage and
edema. In this study, we further investigated whether NKCC1 activity contributes to ischemic damage during either
ischemia or reperfusion. Immunoblotting revealed that expression of
NKCC1 protein was increased following 2-h focal
ischemia in cerebral cortex. A sustained up-regulation of NKCC1 in cortex was detected at 4, 8, 12, and 24 h of reperfusion. An increase in the phosphorylated NKCC1 (NKCC1-p) was found at 4 and 8 h of reperfusion. In striatum, a significant increase in NKCC1 expression occurred between 4 and 24 h of reperfusion and no elevation of NKCC1-p signal was observed. Artificial cerebral spinal fluid (aCSF) or 100 microM
bumetanide in aCSF were continuously microdialyzed into left cortices either 1 h prior to
ischemia plus 2-h
ischemia, or only during 24-h reperfusion.
Infarction volume was significantly decreased in the pre-ischemic
bumetanide-treated group (P<0.05) but not in the post-ischemic treatment group (P>0.05). In addition, pre-ischemic
bumetanide treatment reduced the ipsilateral water content increase by 70% (P<0.05). Inhibition of NKCC1 did not attenuate
poly (ADP-ribose) polymerase cleavage or the number of TUNEL-labeled apoptotic cells in ischemic brains. These results suggest that inhibition of NKCC1 attenuates cytotoxic
edema and necrotic neuronal death during focal
ischemia. Activation of NKCC1 activity plays a role in the early stage of ischemic damage.