Skip to main content
. 2022 Sep 7;42(36):7001–7014. doi: 10.1523/JNEUROSCI.2222-21.2022

Figure 4.

Figure 4.

Astrocytic Mt protects neurons from ICH-like injury by upregulating Mn-SOD and inhibiting ROS overproduction in culture. A, ROS generation, assessed with MitoSOX Red mitochondrial superoxide indicator, by neurons in culture (3.0 × 105 neurons/well) pretreated with ACM or mdACM for 24 h before ICH-like injury for 12 h in vitro (RBC lysates, 3.0 × 107 RBCs/well). The significant changes in ROS generation were assessed by one-way ANOVA/Fisher's LSD test (n = 4 per group), **p < 0.01 (p = 0.0004, CON vs VEH plus ICH-like injury], t value (t = 4.841); **p < 0.01 (p = 0.0011, VEH plus ICH-like injury vs ACM plus ICH-like injury), t value (t = 4.271); **p < 0.01 (p = 0.0002, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 5.41). CON, Control; VEH, vehicle. ACM collected from astrocytes on longer incubation is more effective in preventing ROS generation from neurons under ICH-like injury (Extended Data Fig. 4-1). B, Viability of neurons under ICH-like injury conditions and treatment protocol as described for A. Neuronal viability was measured by LDH assay. Values represent fold change in viabilities versus CON. The significance in neuronal viability was assessed by one-way ANOVA/Fisher's LSD test (n = 4 per group), **p < 0.01 (p < 0.0001, CON vs VEH plus ICH-like injury), t value (t = 5.943); **p < 0.01 (p < 0.0001, VEH plus ICH-like injury vs ACM plus ICH-like injury), t value (t = 9.686); **p < 0.01 (p < 0.0001, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 8.784). C, Viability of neurons (3.0 × 105 neurons/well) that were transfected with 10 nm Mn-SOD-siRNA (SOD2 si) or Scr si for 24 h before exposure to ACM or control media for 24 h and then exposed to ICH-like injury in vitro (RBC lysates, 3.0 × 107 RBCs/well) for 48 h. Values represent fold change of viabilities compared with Scr si group. The significance in neuronal viability was assessed by one-way ANOVA/Fisher's LSD test (n = 18 in Scr si, n = 15 in Scr si plus ICH-like injury, 7 in Scr si plus ACM plus ICH-like injury, 25 in SOD2 si plus ICH-like injury, and 11 in SOD2 si plus ACM plus ICH-like injury), **p < 0.01 (p < 0.0001, Scr si vs Scr si plus ICH-like injury), t value (t = 5.354); *p < 0.05 (p = 0.0232, Scr si plus ICH-like injury vs SOD2 si plus ICH-like injury), t value (t = 2.32); **p < 0.01 (p < 0.0001, Scr si plus ICH-like injury vs Scr si plus ACM plus ICH-like injury), t value (t = 5.963); **p < 0.01 (p = 0.0023, Scr si plus ACM plus ICH-like injury vs SOD2 si plus ACM plus ICH-like injury), t value (t = 3.156); **p < 0.01 (p < 0.0001, SOD2 si plus ICH-like injury vs SOD2 si plus ACM plus ICH-like injury), t value (t = 5.42). D, Representative Western blot image and quantitating bar graph showing Mn-SOD protein levels in cultured neurons treated with ACM or mdACM under ICH-like injury in vitro. The significant changes in Mn-SOD/β-actin protein level were assessed by one-way ANOVA/Fisher's LSD test (n = 5 per group), **p < 0.01 (p = 0.0060, CON vs ICH-like injury), t value (t = 3.166); **p < 0.01 (p = 0.0009, ICH-like injury vs ACM plus ICH-like injury), t value (t = 4.041); **p < 0.01 (p = 0.0008, ACM plus ICH-like injury vs mdACM plus ICH-like injury), t value (t = 4.113). Mn-SOD is present in ACM but not in mdACM, and Mn-SOD level is higher in astrocytes than in neurons, in vitro (Extended Data Fig. 4-2). E, Mn-SOD mRNA levels in cultured neurons treated with ACM or SI plus ACM under ICH-like injury in vitro. SI, STAT3 inhibitor. Neurons were pretreated with ACM or 2 μm SI plus ACM for 24 h, followed by exposure to ICH-like injury for 48 h. Values represent fold change of Mn-SOD mRNA levels compared with CON group. The significant changes in Mn-SOD mRNA level were assessed by one-way ANOVA/Fisher's LSD test (n = 8 per group), **p < 0.01 (p = 0.0032, CON vs ICH-like injury), t value (t = 3.223); **p < 0.01 (p < 0.0001, ICH-like injury vs ACM plus ICH-like injury), t value (t = 12.19); **p < 0.01 (p = 0.0025, ACM plus ICH-like injury vs SI plus ACM plus ICH-like injury), t value (t = 3.316). All data are shown as mean ± SEM.