Prophylactic Zinc and Therapeutic Selenium Administration Increases the Antioxidant Enzyme Activity in the Rat Temporoparietal Cortex and Improves Memory after a Transient Hypoxia-Ischemia

Abstract

In the cerebral hypoxia-ischemia rat model, the prophylactic administration of zinc can cause either cytotoxicity or preconditioning effect, whereas the therapeutic administration of selenium decreases the ischemic damage. Herein, we aimed to explore whether supplementation of low doses of prophylactic zinc and therapeutic selenium could protect from a transient hypoxic-ischemic event. We administrated zinc (0.2 mg/kg of body weight; ip) daily for 14 days before a 10 min common carotid artery occlusion (CCAO). After CCAO, we administrated sodium selenite (6 μg/kg of body weight; ip) daily for 7 days. In the temporoparietal cerebral cortex, we determined nitrites by the Griess method and lipid peroxidation by the Gerard-Monnier assay. qPCR was used to measure mRNA of nitric oxide synthases, antioxidant enzymes, chemokines, and their receptors. We measured the enzymatic activity of SOD and GPx and protein levels of chemokines and their receptors by ELISA. We evaluated long-term memory using the Morris-Water maze test. Our results showed that prophylactic administration of zinc caused a preconditioning effect, decreasing nitrosative/oxidative stress and increasing GPx and SOD expression and activity, as well as eNOS expression. The therapeutic administration of selenium maintained this preconditioning effect up to the late phase of hypoxia-ischemia. Ccl2, Ccr2, Cxcl12, and Cxcr4 were upregulated, and long-term memory was improved. Pyknotic cells were decreased suggesting prevention of neuronal cell death. Our results show that the prophylactic zinc and therapeutic selenium administration induces effective neuroprotection in the early and late phases after CCAO.

Figure 1

The combined treatment with zinc and selenium decreases the CCAO-induced nitrosative-oxidative stress in the temporoparietal cortex. (a) Nitrosative stress and (b) lipid peroxidation. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO; Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control untreated group; nv: normalized values. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared to the control untreated group. ^†P < 0.05, Student's t-test when compared with the respective CCAO group.

Figure 2

Effect of the combined treatment with zinc and selenium on nitric oxide synthase transcription and translation in the temporoparietal cortex of ischemic rats. (a), (c), and (e) mRNA levels; (b), (d), and (f) protein levels. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO: Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control group. nv: normalized values. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared to the control untreated group; ^†P < 0.05, Student's t-test when compared with the respective CCAO group.

Figure 3

The combined treatment with zinc and selenium increased the enzymatic activity and transcription of glutathione peroxidase in the late phase of hypoxia-ischemia in the rat. (a) Enzymatic activity of GPx and (b) mRNA levels of GPx4. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO; Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control group. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared to the control untreated group; ^†P < 0.05, Student's t-test when compared with the respective CCAO group.

Figure 4

Differential effect of the combined treatment with zinc and selenium on the enzymatic activity and transcription of superoxide dismutase in the rat hypoxia-ischemia model. (a) Enzymatic activity of SOD, (b) mRNA levels of Sod1, (c) mRNA levels of Sod2, and (d) mRNA levels of Sod3. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO; Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control group. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared to the control untreated group; ^†P < 0.05, Student's t-test when compared with the CCAO group.

Figure 5

Differential effect of the combined treatment with zinc and selenium on chemokine and receptor transcription in the rat hypoxia-ischemia model. (a), (c), and (e) Chemokines levels; (b), (d), (f) receptors of the respective chemokines. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO; Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control group. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared to the control untreated group; ^†P < 0.05, Student's t-test when compared with the respective CCAO group.

Figure 6

The administration of the combined treatment with zinc and selenium modifies the protein levels of chemokines and receptors in the rat hypoxia-ischemia model. (a), (c), and (e) Chemokine levels, and (b), (d), (f) receptors of the respective chemokines. CCAO: common carotid artery occlusion for 10 min; Zn + CCAO: chronic zinc administration before CCAO. Zn + CCAO + Se: chronic zinc administration before CCAO followed by selenium administration. The values were normalized against the control group. nv: normalized values. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared with the untreated control group; ^†P < 0.05, Student's t-test when compared with the respective CCAO group.

Figure 7

The administration of the combined treatment with zinc and selenium decreases the number of pyknotic cells in the temporoparietal cortex of hypoxia-ischemic rats. Representative micrographs with hematoxylin-eosin staining. The headings indicate the different experimental conditions. (g) Counting of pyknotic cells in the control groups treated with zinc or zinc + Se. (h) Counting of pyknotic cells in the groups with CCAO and treated with zinc (zinc) or the combined treatment with zinc (zinc + CCAO) and selenium (zinc + CCAO + Se). Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way ANOVA with post hoc Dunnett's test when compared with the untreated control group; ^†P < 0.05, Student's t-test when compared with the CCAO group.

Figure 8

The administration of the combined treatment with zinc and selenium improves information consolidation in the hypoxia-ischemia model in rats. Control = untreated rats; Zn = zinc; CCAO = common carotid artery occlusion; Zn + CCAO = chronic zinc administration before CCAO; Zn + CCAO + Se = chronic zinc administration before CCAO followed by selenium (Se) administration. Each value represents mean ± SEM of 5 independent experiments made in triplicate. ^∗P < 0.05, one-way Kruskal-Wallis analysis of variance with post hoc Dunnett's test when compared with the untreated control group; ^†P < 0.05, Mann-Whitney U test when compared with the CCAO group.