Increased NKCC1 and decreased KCC2 after TBI.
a,
b Immunohistochemical staining of NKCC1 (
a1,
b1), NeuN (
a2,
b2), and the merged image (
a3,
b3) are illustrated to show neuronal NKCC1 in layers
i–
vi of cortex, at 3 days following sham (
a1–
a3) and TBI (
b1–
b3) mice. In
c, an example of a western blot assessing NKCC1 in sham and TBI mice at 3 h, 1, 3, and 7 days post-FPI. Quantitative analysis of NeuN
+/NKCC1
+ cells (
d) revealed a significant increase after by 1 day post-TBI and persisting until at least 7 days after TBI. The counts represent number of NKCC1
+/NeuN
+ cells per 25,000 µm
2. Consistent with this finding, western blot analysis (
e) of NKCC1 in cortical tissue revealed a significant increase (
F (4,15) = 4.868,
P < 0.05, one-way ANOVA) at 3 days post-TBI. It is pertinent to note that we also present qPCR analysis in both cortex and hippocampus, and immunohistochemistry in cortex, showing that the elevation of NKCC1 is bilateral (see Supplementary Figs.
1 and
2). In
f–
g, KCC2 and NeuN double-labeling is shown to depict a decrease in KCC2 following TBI (
g1–
g3) compared with shams (
f1–
f3).
h Western blot assessing KCC2 in sham and TBI mice at 3 h, 1, 3, and 7 days post-FPI. Quantitative analysis of the NeuN
+/KCC2
+ cells (
i) demonstrated that a significant decrease by 3 days after TBI. The counts represent number of KCC2
+/NeuN
+ cells per 25,000 µm
2 of primary motor and somatosensory cortex. Western blot analysis (
j) revealed that protein levels of KCC2 were significantly decreased (
F (4,15) = 1.716,
P < 0.05) in cortex by 3 days after TBI