Prophylactic Subacute Administration of Zinc Increases CCL2, CCR2, FGF2, and IGF-1 Expression and Prevents the Long-Term Memory Loss in a Rat Model of Cerebral Hypoxia-Ischemia

Abstract

Prophylactic subacute administration of zinc decreases lipoperoxidation and cell death following a transient cerebral hypoxia-ischemia, thus suggesting neuroprotective and preconditioning effects. Chemokines and growth factors are also involved in the neuroprotective effect in hypoxia-ischemia. We explored whether zinc prevents the cerebral cortex-hippocampus injury through regulation of CCL2, CCR2, FGF2, and IGF-1 expression following a 10 min of common carotid artery occlusion (CCAO). Male rats were grouped as follows: (1) Zn96h, rats injected with ZnCl2 (one dose every 24 h during four days); (2) Zn96h + CCAO, rats treated with ZnCl2 before CCAO; (3) CCAO, rats with CCAO only; (4) Sham group, rats with mock CCAO; and (5) untreated rats. The cerebral cortex-hippocampus was dissected at different times before and after CCAO. CCL2/CCR2, FGF2, and IGF-1 expression was assessed by RT-PCR and ELISA. Learning in Morris Water Maze was achieved by daily training during 5 days. Long-term memory was evaluated on day 7 after learning. Subacute administration of zinc increased expression of CCL2, CCR2, FGF2, and IGF-1 in the early and late phases of postreperfusion and prevented the CCAO-induced memory loss in the rat. These results might be explained by the induction of neural plasticity because of the expression of CCL2 and growth factors.

Figure 1

Effect of subacute administration of zinc on CCL2 expression in cerebral cortex-hippocampus of the rat. RT-PCR was used to determine mRNA levels of CCL2 (323 bp) and GA3PDH (420 bp). ((a) and (b)) Showing representative photographs of ethidium-bromide-stained RT-PCR products fractionated on 2% agarose gel and the respective densitometry analysis. ((c) and (d)) Showing the CCL2 protein levels using ELISA. Each value represents the mean ± SEM of 5 independent experiments made in triplicate. (1) Zn96h, rats injected with ZnCl₂ (one dose every 24 h during 4 days). (2) Zn96h + CCAO, rats treated with zinc before 10 min of common carotid artery occlusion (CCAO). (3) CCAO, rats with CCAO only. (4) Sham group, rats with mock CCAO. (5) Untreated rats. ∗, significant when compared with the control group; ANOVA test and post hoc Dunnett's test. †, significant when compared between groups; unpaired Student's t-test. P < 0.05.

Figure 2

The subacute administration effect of zinc on levels of CCR2 expression after hypoxia-ischemia in rats. RT-PCR was used to determine mRNA levels of CCR2 (311 bp) and GA3PDH (420 bp). ((a) and (b)) Showing representative photographs of ethidium-bromide-stained RT-PCR products fractionated on 2% agarose gel and the respective densitometry analysis. ((c) and (d)) Showing the CCR2 protein levels measured using ELISA. Each value represents the mean ± SEM of 5 independent experiments in triplicate. (1) Zn96h, rats injected with ZnCl₂ (one dose every 24 h during 4 days). (2) Zn96h + CCAO, rats treated with zinc before 10 min of common carotid artery occlusion (CCAO). (3) CCAO, rats with CCAO only. (4) Sham group, rats with mock CCAO. (5) Untreated rats. ∗, significant when compared with the control group; ANOVA test and post hoc Dunnett's test. †, significant when compared between groups; unpaired Student's t-test. P < 0.05.

Figure 3

Effect of common carotid artery occlusion on the immunoreactivity against CCR2. Representative micrographs of CCR2 immunofluorescence ((a) to (o)) using a rabbit antibody against CCR2 and a goat antibody anti-rabbit IgG conjugated with fluorescein isothiocyanate (green color). The cerebral region was identified by propidium iodide (red color) counterstaining. CA, Cornu Ammonis, and DG, dentate gyrus, of the hippocampus. LV, layer V of the cerebral cortex. Plexus, choroid plexus. Values are the mean ± SEM from 3 rats in each experimental condition.

Figure 4

Effect of the subacute administration of zinc on CCR2 immunoreactivity after CCAO. Representative micrographs of CCR2 immunofluorescence ((a) to (o)) using a rabbit antibody against CCR2 and a goat antibody anti-rabbit IgG conjugated with fluorescein isothiocyanate (green color). The cerebral region was identified with propidium iodide (red color) counterstaining. Graphs ((p), (q), (r), (s), and (t)) show the normalized values of CCR2-IR with respect to control IR for CCAO (Figures 3(a) to 3(o)) and subacute administration of zinc ((a) to (o)). The IR was measured using ImageJ 1.45 of the National Institute of Health. CA, Cornu Ammonis, and DG, dentate gyrus, of the hippocampus. LV, layer V of the cerebral cortex. Plexus, choroid plexus. Values are expressed as the mean ± SEM from 3 rats for each experimental condition. ∗, significant when compared with the control group; ANOVA test and post hoc Dunnett's test. †, significant when compared between groups; unpaired Student's t-test. P < 0.05.

Figure 5

The subacute administration effect of zinc on FGF2 expression levels after hypoxia-ischemia in the rat. RT-PCR was used to determine mRNA levels of FGF2 (260 bp) and GA3PDH (420 bp). ((a) and (b)) Showing representative photographs of ethidium-bromide-stained RT-PCR products fractionated on 2% agarose gel and the respective densitometry analysis. ((c) and (d)) Showing the FGF2 protein levels measured using ELISA. Each value represents the mean ± SEM of 5 independent experiments made in triplicate. (1) Zn96h, rats injected with ZnCl₂ (one dose every 24 h during 4 days). (2) Zn96h + CCAO, rats treated with zinc before 10 min common carotid artery occlusion (CCAO). (3) CCAO, rats with CCAO only. (4) Sham group, rats with mock CCAO. (5) Untreated rats. ∗, significant when compared with the control group; ANOVA test and post hoc Dunnett's test. †, significant when compared between groups; unpaired Student's t-test. P < 0.05.

Figure 6

The subacute administration effect of zinc on levels of IGF-1 expression after hypoxia-ischemia in rats. RT-PCR was used to determine mRNA levels of IGF-1 (381 bp) and GA3PDH (420 bp). ((a) and (b)) Showing representative photographs of ethidium-bromide-stained RT-PCR products fractionated on 2% agarose gel and the respective densitometry analysis. ((c) and (d)) Showing the IGF-1 protein levels measured using ELISA. Each value represents the mean ± SEM of 5 independent experiments in triplicate. (1) Zn96h, rats injected with ZnCl₂ (one dose every 24 h during 4 days). (2) Zn96h + CCAO, rats treated with zinc before 10 min of common carotid artery occlusion (CCAO). (3) CCAO, rats with CCAO only. (4) Sham group, rats with mock CCAO. (5) Untreated rats. ∗, significant when compared with the control group; ANOVA test and post hoc Dunnett's test. †, significant when compared between groups; unpaired Student's t-test. P < 0.05.

Figure 7

The effect of subacute administration of zinc on learning and long-term memory after hypoxia-ischemia in rats. ((a) and (b)) Graphs showing the latency to reach the escape platform in the fourth event (eastern quadrant) of a daily evaluation for five days in the Morris Water Maze (n = 10 rats per group). (c) Graphs showing the latency determined on day 7 after the learning training, that is, on day 12 after reperfusion (n = 5 rats per group). (d) Graphs showing the number of times in southeast (# Time in SE) at which rats pass by the platform location. Evaluations were made on day 7 after the learning training. The values are the mean ± SEM. ∗, significant when compared with the control group ANOVA test and post hoc Dunnett's test, P < 0.05. †, significant when compared with the CCAO group; unpaired Student's t-test. P < 0.05.