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. 2016 Apr 20;36(16):4443–4456. doi: 10.1523/JNEUROSCI.3691-15.2016

Figure 6.

Figure 6.

Effects of l-lactate on survival and oxidative stress of ARALAR-deficient primary neuronal cultures challenged with exogenous glutamate. A, Increase in glutamate released to the neuronal culture media, 24 h after treatment in conditions of 10 mm l-lactate under glucose deprivation in WT and aralar-KO cultures. B, Cellular viability was evaluated 24 h after maintaining cultures for 6 h in the previously mentioned condition. Data are calculated as an increase in percentage of dead cells, compared with control condition with 5.0 mm glucose using the calcein-AM/propidium iodide method. C, Cortical cell cultures were pretreated with 10 mm l-lactate or 2 mm pyruvate 30 min before 5 min exposure to 25 μm glutamate and maintained for 24 h in the presence of l-lactate or pyruvate. Data are expressed as percentage of dead cells, PI/(CL+PI), under control treatment (Ctr), glutamate exposure (Glu 25), glutamate plus l-lactate (Glu 25 + Lac), or glutamate plus pyruvate (Glu25 + pyr). Data are mean ± SEM from three independent experiments. *p ≤ 0.05 (one-way ANOVA followed by Student-Newman-Keuls t test). **p ≤ 0.01 (one-way ANOVA followed by Student-Newman-Keuls t test). D–G, Detection of 8-OHdG immunostaining in mitochondrial and nuclear DNA induced by 100 μm glutamate exposure for 30 min alone or in the presence of 10 mm l-lactate. Photomicrographs representative for control (CTR), glutamate (Glu), or glutamate plus l-lactate (Glu + Lac) conditions are shown in control (D) and aralar-KO (F). Coimmunostaining was performed using TOPRO-3 (nuclear marker) or Mn-SOD (mitochondrial marker) and quantified in E and G, respectively, to elucidate the primary DNA damage induced in our experimental conditions. Integrated and average fluorescence intensity was calculated using the MetaMorph program. n = 177–349, or 218–304 cells per condition, in WT and aralar-KO cultures, respectively. *p ≤ 0.05 (ANOVA followed by followed by Student-Newman-Keuls t test). **p ≤ 0.01 (ANOVA followed by followed by Student-Newman-Keuls t test). ***p ≤ 0.001 (ANOVA followed by followed by Student-Newman-Keuls t test). n.s, Not significant.