Subacute zinc administration and L-NAME caused an increase of NO, zinc, lipoperoxidation, and caspase-3 during a cerebral hypoxia-ischemia process in the rat

Abstract

Zinc or L-NAME administration has been shown to be protector agents, decreasing oxidative stress and cell death. However, the treatment with zinc and L-NAME by intraperitoneal injection has not been studied. The aim of our work was to study the effect of zinc and L-NAME administration on nitrosative stress and cell death. Male Wistar rats were treated with ZnCl2 (2.5 mg/kg each 24 h, for 4 days) and N-ω-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg) on the day 5 (1 hour before a common carotid-artery occlusion (CCAO)). The temporoparietal cortex and hippocampus were dissected, and zinc, nitrites, and lipoperoxidation were assayed at different times. Cell death was assayed by histopathology using hematoxylin-eosin staining and caspase-3 active by immunostaining. The subacute administration of zinc before CCAO decreases the levels of zinc, nitrites, lipoperoxidation, and cell death in the late phase of the ischemia. L-NAME administration in the rats treated with zinc showed an increase of zinc levels in the early phase and increase of zinc, nitrites, and lipoperoxidation levels, cell death by necrosis, and the apoptosis in the late phase. These results suggest that the use of these two therapeutic strategies increased the injury caused by the CCAO, unlike the alone administration of zinc.

Figure 1

Effect of subacute administration of zinc and L-NAME on zinc levels during a cerebral hypoxia-ischemic process. The zinc levels were assayed by the Johnson method described elsewhere [18, 25]. Each value is the mean ± SE of 5 independent experiments made in triplicate. Zn96h + CCAO: preventive subacute administration of zinc (2.5 mg/kg intraperitoneal each 24 hours during 4 days) and common carotid-artery occlusion (CCAO) for 10 min. Zn96h + L-NM + CCAO: rats treated with zinc in the presence of an inhibitor of nitric oxide synthase (L-NAME) one hour before the CCAO. *P < 0.05, an ANOVA test and a post-hoc Dunnet test to compare with the control group, and ^† P < 0.05, unpaired Student's t-test to compare between groups.

Figure 2

Subacute administration of zinc and L-NAME on nitrite levels during a cerebral hypoxia-ischemic process. The nitrite levels were assayed by the Griess method described elsewhere [18, 25]. Each value is the mean ± SE of 5 independent experiments made in triplicate. Zn96h + CCAO: preventive subacute administration of zinc (2.5 mg/kg each 24 hours during 4 days) and a common carotid-artery occlusion (CCAO) for 10 min. Zn96h + L-NM + CCAO: rats treated with zinc in presence of an inhibitor of nitric oxide synthase (L-NAME) one-hour before CCAO. *P < 0.05, an ANOVA test and a post-hoc Dunnet test to compare with control group, and ^† P < 0.05, unpaired Student's t-test to compare between groups.

Figure 3

Subacute administration of zinc and L-NAME on lipoperoxidation levels during a cerebral hypoxia-ischemic process. Malonyldialdehyde (MDA) and 4-hydroxyalkanal (4-HEA) concentrations measured by using the method described elsewhere [18, 25] were used as biomarkers of lipoperoxidation. Each value represents the mean ± SE of 5 independent experiments made in triplicate. Zn96h + CCAO: preventive subacute administration of zinc (2.5 mg/kg each 24 hours for 4 days) and common carotid-artery occlusion (CCAO) for 10 min. Zn96h + L-NM + CCAO: rats treated with zinc in the presence of an inhibitor of nitric oxide synthase (L-NAME) one-hour before the CCAO. *P < 0.05, an ANOVA test and a post-hoc Dunnet test to compare with the control group, and ^† P < 0.05, unpaired Student's t-test to compare between groups.

Figure 4

Hematoxylin-eosin staining in slides of the temporoparietal cortex and hippocampus in rats treated with zinc in the presence or absence of L-NAME. Paraffin-embedded tissue sections of 3 μm were stained with hematoxylin and eosin. The Zn96h + CCAO: preventive subacute administration of zinc (2.5 mg/kg each 24 hours for 4 days) and a common carotid-artery occlusion (CCAO) for 10 min. Zn96h + L-NM + CCAO: rats treated with zinc in the presence of an inhibitor of nitric oxide synthase (L-NAME) one hour before a CCAO. CA1 (a–e), CA3 (f–j) regions and dentate gyrus (DG: (k–o)) of hippocampus and LV, layer V of cerebral cortex (p–t). Apoptosis cell (dark arrowhead), necrosis (clear arrowhead), and branched cells (arrow).

Figure 5

Immunohistochemistry against caspase-3 and Nissl counterstaining in slides of the hippocampus and temporoparietal cortex in zinc-treated rats in the presence or absence of L-NAME. The labels at the left side of the micrographs are cerebral regions. The immunostaining against cleaved caspase-3 is shown by the dark marks (dark arrowhead), and the Nissl stain appears in blue and pale cell (clear arrowhead). Zn96h + CCAO: preventive subacute administration of zinc (2.5 mg/kg each 24 hours for 4 days) and the common carotid-artery occlusion (CCAO) for 10 min. Zn96h + L-NAME + CCAO: rats treated with zinc in presence of an inhibitor of nitric oxide synthase (L-NAME) one-hour before the CCAO. DG: dentate gyrus of hippocampus; LV: layer V of cerebral cortex.