Neuroglobin protects neurons against oxidative stress in global ischemia

Abstract

Neuroglobin (Ngb) is a recently discovered globin that affords protection against hypoxic/ischemic-induced cell injury in brain. Hypoxic/ischemic injury is associated with accumulation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In previous studies, we found that Ngb has antioxidative properties, and protects PC-12 cells against hypoxia- and β-amyloid-induced cell death. To further delineate the potential role of Ngb in protection against cerebral ischemia-reperfusion injury in vivo, we developed a transgenic mouse line that overexpresses Ngb. Hippocampal ischemia-reperfusion injury was induced by a 10-minute bilateral occlusion of the common carotid arteries, and the animal brains were assessed 3 days later. CA1 neural injury was determined by cresyl violet staining. Lipid peroxidation was assessed using a malonyldialdehyde assay kit, whereas ROS/RNS accumulation was determined by Het staining in the CA1 hippocampal region. Hippocampal Ngb mRNA and protein expressions were assessed by reverse transcriptase-PCR and western blotting, respectively. Neuroglobin was successfully overexpressed in the hippocampus of Ngb transgenic mice. After ischemia-reperfusion, CA1 ROS/RNS production and lipid peroxidation were markedly decreased in Ngb transgenic mice compared with wild-type mice. Furthermore, CA1 neuronal injury was also markedly reduced. Thus, Ngb may confer protection against ischemia-reperfusion injury in the brain through its intrinsic antioxidant properties.

Figure 1

Ngb mRNA and protein expressions in the hippocampus of Ngb transgenic mice. The hippocampal tissues of Ngb transgenic mice or wild-type mice (male, 10 weeks old) were harvested and subjected to either Ngb mRNA and protein analyses or to immunohistochemistry. (A) Ngb mRNA expression in the hippocampus of Ngb transgenic and wild-type mice. Data were expressed as a percentage of wild type (n=8 per group, ^*P<0.01 versus wild type). (B) Ngb protein expression in the hippocampal tissue of Ngb transgenic and wild-type mice. Data are expressed as a percentage of wild type (n=8 per group). (C) Western blot images of Ngb and β-actin protein expressions in the hippocampal tissues of Ngb transgenic and wild-type mice. (D and E) Immunohistochemical staining of Ngb and NeuN in the hippocampus of Ngb transgenic and wild type mouse. NeuN, neuronal nuclei; NgB, neuroglobin.

Figure 2

Effect of Ngb overexpression on the SOD, GPX, and catalase activity in the hippocampus. Hippocampal tissues from Ngb transgenic mice or wild-type mice (male, 10 weeks, n=8 in each group) were harvested (A) before or (B) 1 day after surgery and subjected to assessment of the enzymatic activities of SOD, GPX, and catalase. Data are expressed as the percentage of wild type (n=8 per group, P>0.05). NgB, neuroglobin; SOD, superoxide dismutase.

Figure 3

Effect of Ngb overexpression on ischemia–reperfusion-induced lipid peroxidation and nitrotyrosine formation. After 10 minutes of ischemia and 3-day recovery, the hippocampal tissues were harvested in Ngb transgenic mice or wild-type mice (male, 10 weeks, n=8 in each group), and subjected to assessment of lipid peroxidation and nitrotyrosine formation. (A) Effect of Ngb overexpression on ischemia–reperfusion induced MDA formation. Data were expressed as a percentage of sham control (n=8 per group, ^*P<0.01 versus sham control, ^#P<0.01 versus wild type). (B) Effect of Ngb overexpression on ischemia–reperfusion induced nitrotyrosine formation. Data are expressed as a percentage of sham control (n=6 per group). MDA, malonyldialdehyde; NgB, neuroglobin.

Figure 4

Effect of Ngb overexpression on ischemia–reperfusion-induced ROS/RNS production and neuronal injury. After 10 minutes ischemia and 1- or 3-day recovery periods, Ngb transgenic mice or wild-type mice (male, 10 weeks old) were deeply anesthetized and perfused intracardially with 4% phosphate-buffered paraformaldehyde. Serial hippocampal sections were obtained and subjected to Het staining and to quantitative analysis of neuronal damage. (A) Het fluorescence in the hippocampus after ischemia–reperfusion. Data are expressed as a percentage of sham control (n=9 per group, ^*P<0.01 versus sham control, ^#P<0.01 versus wild type). (B) Cresyl violet staining of the hippocampus after ischemia–reperfusion. (C) Quantitative analysis of neuronal damage in the CA1 region of the hippocampus after ischemia–reperfusion. Data are expressed as the percentage of damaged neurons from the total number of neurons counted (n=9 per group, Mann–Whitney U-test). NgB, neuroglobin; RNS, reactive nitrogen species; ROS, reacticve oxygen species.