The effect of hypertonic saline on notch signaling pathway in experimentally induced cerebral ischemic rats

Abstract

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats. Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group (NS group), 10% hypertonic saline group (10%HS group), the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion (MCAO). After 2 hours of MCAO, the rats were through reperfusion for 24 h. In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 mL/h) and 10% HS (0.3 mL/h) by tail vein for 24 h. Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain (NICD). All data was analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P<0.05. Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group; the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group. RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 1.000±0.076; ischemia group: 2.203±0.283; NS group: 1.616±0.185; P<0.01); however, it was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 2.203±0.283; NS group: 1.616±0.185; 10%HS group: 1.202±0.177; P<0.05). Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.290±0.079; ischemia group: 0.750±0.029; NS group: 0.765±0.182; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group (ischemia group: 0.750±0.029; NS group: 0.765±0.182; 10%HS group: 0.390±0.195; P<0.05). The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.401±0.196; ischemia group: 0.906±0.359; NS group: 0.847±0.153; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 0.906±0.359; NS group: 0.847±0.153; 10%HS group: 0.561±0.165; P<0.05). Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats. Key words: Hypertonic saline; Normal saline; Cerebral ischemia; Middle cerebral artery occlusion (MCAO); Microglia; Notch signaling; Notch1; Notch receptor domain (NICD)