Experimental Design

All experiments were performed on DIV 5–7 astroglial cells grown up in culture medium containing 10% of FBS. After removal of medium, cells were incubated at 37°C with fresh serum-free culture medium in the absence or presence of the test substances for 24 h. Previous data indicated that incubation of astrocytes during 24 h with 50 µM H₂O₂ induces about 40% of cell death (36). Thus, a dose of 50 µM H₂O₂ was used in the present study to evaluate the effect of Hb on astrocyte survival. On the basis of the results shown in Figures ​Figures1,1, ​,22 and ​and3A,3A, a concentration of 10⁻⁹ M Hb, which prevents the deleterious effects of H₂O₂, was used in all subsequent experiments. It has been previously demonstrated that 2 × 10⁻⁵ M H89, 10⁻⁶ M chelerythrine, and 10⁻⁶ M U0126 blocked the protective effects of some neuropeptides on cultured astrocytes (35, 37). Thus these concentrations were also used in the present study.

Glioprotective effect of Hb on H₂O₂-induced cell death. (A) Cells were co-incubated for 24 h with medium alone () or with H₂O₂ (50 µM) in the absence () or presence of graded concentration of Hb (10⁻¹² to 10⁻⁶ M, ). Cell survival was quantified by measuring FDA fluorescence intensity, and the results are expressed as percentages of the control. Data are means ± SEM of four independent experiments. ANOVA followed by Bonferroni’s test ***p < 0.001; NS, not statistically different from control cells (absence of H₂O₂ and absence of Hb). ^##p < 0.01; ^###p < 0.001; ns, not statistically different vs. H₂O₂-treated cells. (B) Typical phase-contrast images illustrating the effect of Hb on H₂O₂-induced morphological changes in cultured rat astrocytes. Cells were incubated for 24 h with medium alone (a), or 50 µM H₂O₂ in the absence (b), or presence of 10⁻¹² M Hb (c), or 10⁻⁹ M Hb (d). Scale bar 100 µm.

Effect of Hb on H₂O₂-induced ROS and NO intracellular accumulations. Cells were co-incubated for 24 h with medium alone () or with H₂O₂ (50 µM) in the absence () or presence of graded concentration of Hb (10⁻¹² to 10⁻⁶ M, ). (A) Cellular ROS level was quantified by measurement of DCF fluorescence intensity. (B) Cellular NO level was quantified by measurement of DAF fluorescence intensity. Inset, effect of graded concentration of Hb (10⁻¹² to 10⁻⁶ M; ) on intracellular ROS accumulation (A) and NO formation (B). The results are expressed as percentages of the controls. Data are means ±SEM of three independent experiments. ANOVA followed by Bonferroni’s test *p < 0.05; **p < 0.01; ***p < 0.001; NS, not statistically different from control cells. ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ns, not statistically different vs. H₂O₂-treated cells.

Effect of Hb on H₂O₂-induced alteration of mitochondrial membrane potential and caspase-3 activation in cultured astrocytes. Cells were co-incubated for 24 h with medium alone () or with H₂O₂ (50 µM) in the absence () or presence of graded concentrations of Hb (10⁻¹² to 10⁻⁶ M, ). (A) Mitochondrial transmembrane potential was assessed by using the JC-10 probe, and the ratio of fluorescence emissions 610/530 nm was measured as an index of mitochondrial activity. (B) Caspase 3 activity was measured by caspase substrate, Z-DEVDRhodamine 110, fluorescence. The results are expressed as percentage of controls. Data are means ±SEM of three independent experiments. ANOVA followed by Bonferroni’s test. **p < 0.01; ns, not statistically different from control cells. ^##p < 0.01; ^###p < 0.001; ns, not statistically different vs. H₂O₂-treated cells.