Na+-Dependent Chloride Transporter (NKCC1)-Null Mice Exhibit Less Gray and White Matter Damage after Focal Cerebral Ischemia

Abstract

We previously demonstrated that pharmacological inhibition of Na+−K+−Cl− cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (˜85 mm3) in NKCC1 wild-type (NKCC1+/+) mice. Infarction volume in NKCC1−/− mice was reduced by ˜30% to 46%. Heterozygous mutant (NKCC1+/–) mice showed ˜28% reduction in infarction ( P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1+/+ mice. In contrast, NKCC1+/– and NKCC1−/− mice exhibited ˜50% less edema ( P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1+/+ mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1−/− mice exhibited significantly less APP accumulation ( P<0.05). Oxygen-glucose deprivation (OGD) induced ˜67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1+/+ cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1−/− neurons ( P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1+/+ neurons and astrocytes ( P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.