Figure 10

HS may directly down-regulate NKCC1 mRNA and protein expression in primary astrocytes under hypoxic condition. Bar graph A shows that NKCC1 mRNA expression in primary astrocytes decreased significantly (*P < 0.05) following treatment with HS or bumetanide under hypoxic conditions. Panels B and C show the optical density of NKCC1 protein is significantly increased following hypoxic exposure as compared with control group (#P < 0.05); however, NKCC1 protein expression is markedly reduced (*P < 0.05) after treatment with HS or bumetanide (a specific NKCC1 inhibitor). Immunofluorescence images of astrocytes showing the expression of GFAP-labeled astrocytes (D, G, J, M, red), co-labeled with NKCC1 (E, H, K, N, green). The co-localized expression of GFAP and NKCC1 can be seen in panels F, I , L and O. Note NKCC1 expression is down-regulated in astrocytes after treatment with 100 mM HS (K) as compared with hypoxia group cells (H). ns: non-significant, P > 0.05. Scale bars: (D-L), 20 μm. GFAP, glial fibrillary acidic protein; HS, hypertonic saline; NKCC1, Na-K-Cl Cotransporter 1.