Targeted Disruption of Na⁺/Ca²⁺ Exchanger 3 (NCX3) Gene Leads to a Worsening of Ischemic Brain Damage

Effect of ncx3 ablation on brain ischemia, neuronal survival under hypoxic conditions, [Ca²⁺]_i, and I_NCX. A, Effect of ncx3 knocking-out on infarct volume in C57BL/6 wild-type and congenic ncx3+/+, ncx3+/−, and ncx3−/− mice, respectively, subjected to tMCAo. Each column represents the mean ± SEM of the percentage of the infarct volume compared with the ipsilateral hemisphere. Ischemic mice were killed 24 h after tMCAo. *p < 0.05 versus C57BL/6 wild-type and congenic ncx3+/+ ischemic mice. n = 9–18 animals for each column. A representative brain slice from each ischemic experimental group is shown on the top of each column. B, Effect of NCX3 knock-out on survival of cortical neurons exposed to OGD plus reoxygenation. Left, Cell death quantification was performed by PI and fluorescein dyes in 7–10 DIV embryonic cortical neurons obtained from ncx3+/+, ncx3+/−, and ncx3−/− mice exposed to 3 h of OGD followed by 21 h of reoxygenation or to 24 h of normoxia. At the end of the experiments, cells were stained with PI and fluorescein, and images were acquired as reported in Materials and Methods. The data are reported as percentage of cell death occurring in each group compared with their respective normoxic cells. In normoxic conditions, ncx3+/+, ncx3+/−, and ncx3−/− cortical neurons showed similar levels of cell death (∼15%). *p < 0.05 versus respective normoxic neurons; **p < 0.05 versus respective normoxic neurons and versus ncx3+/+ group. Each bar represents the mean ± SEM of three different experimental values studied in three independent experimental sessions. Right, The bar graph represents basal fura-2 AM-detected [Ca²⁺]_i in 7–10 DIV cortical neurons from ncx3+/+, ncx3+/−, and ncx3−/− mice. *p < 0.05 versus ncx3+/+. fura-2 AM fluorescence intensity was measured every 3 s for 5 min. The values obtained in this interval were averaged for each cortical neuron. Each bar represents the mean ± SEM of at least 70 cortical neurons in three different experimental sessions. C, D, Effect of ncx3 knocking-out on I_NCX recorded in the reverse mode of operation in 7–10 DIV embryonic cortical neurons. C, I_NCX superimposed traces recorded from ncx3+/+ (left) and ncx3−/− (right) cortical neurons under normoxic conditions (black traces) and after 3 h of OGD (gray traces). D, I_NCX quantification expressed as pA/pF, in ncx3+/+ and ncx3−/− cortical neurons in control conditions and after 3 h of OGD. The values are expressed as mean ± SEM of current densities recorded from 20 cells in each experimental group obtained from three independent experimental sessions. *p < 0.05 versus ncx3+/+ normoxic neurons; **p < 0.05 versus all the other experimental groups.