Prophylactic zinc and therapeutic selenium administration in adult rats prevents long-term cognitive and behavioral sequelae by a transient ischemic attack

Abstract

The transient hypoxic-ischemic attack, also known as a minor stroke, can result in long-term neurological issues such as memory loss, depression, and anxiety due to an increase in nitrosative stress. The individual or combined administration of chronic prophylactic zinc and therapeutic selenium is known to reduce nitrosative stress in the first seven days post-reperfusion and, due to an antioxidant effect, prevent cell death. Besides, zinc or selenium, individually administered, also causes antidepressant and anxiolytic effects. Therefore, this work evaluated whether combining zinc and selenium could prevent stroke-elicited cognition and behavior deficits after 30 days post-reperfusion. Accordingly, we assessed the expression of growth factors at 7 days post-reperfusion, a four-time course of memory (from 7 to 28 days post-learning test), and cell proliferation, depression, and anxiety-like behavior at 30 days post-reperfusion. Male Wistar rats with a weight between 190 and 240 g) were treated with chronic prophylactic zinc administration with a concentration of 0.2 mg/kg for 15 days before common carotid artery occlusion (10 min) and then with therapeutic selenium (6 μg/kg) for 7 days post-reperfusion. Compared with individual administrations, the administration combined of prophylactic zinc and therapeutic selenium decreased astrogliosis, increased growth factor expression, and improved cell proliferation and survival in two regions, the hippocampus, and cerebral cortex. These effects prevented memory loss, depression, and anxiety-like behaviors. In conclusion, these results demonstrate that the prophylactic zinc administration combined with therapeutic selenium can reduce the long-term sequelae caused by the transient ischemic attack. Significance statement. A minor stroke caused by a transient ischemic attack can result in psychomotor sequelae that affect not only the living conditions of patients and their families but also the economy. The incidence of these micro-events among young people has increased in the world. Nonetheless, there is no deep understanding of how this population group responds to regular treatments (Ekker and et al., 2018) [1]. On the basis that zinc and selenium have antioxidant, anti-inflammatory, and regenerative properties in stroke animal models, our work explored whether the chronic combined administration of prophylactic zinc and therapeutic selenium could prevent neurological sequelae in the long term in a stroke rat model of unilateral common carotid artery occlusion (CCAO) by 10-min. Our results showed that this combined treatment provided a long-term neuroprotective effect by decreasing astrogliosis, memory loss, anxiety, and depression-like behavior.

Keywords: Depression-anxiety; Growth factors; Nitrosative stress; Reactive astrogliosis; Selenium; Zinc.

Fig. 1

Schematic diagram of the experimental protocol.

Fig. 2

Prophylactic zinc and therapeutic selenium administrations improved spatial learning and memory. A) Five-day learning test in the Morris water maze. Memory was evaluated over days post-learning, as shown in the headings (B, C, D, and E). Values represent the mean ± SEM of 6 rats and their statistical differences were analyzed using a two-way ANOVA. Statistical significance was indicated as *P < 0.05, or **P < 0.01, when compared to the Control group, and †P < 0.05 and ††P < 0.01, vs. the CCAO group.

Fig. 3

Prophylactic zinc and therapeutic selenium administrations prevented anxiety and depression-like behavior evaluated in elevated plus-maze (A and B) and recovered motor activity (C and D). Values represent the mean ± SEM of 6 rats. Statistical significance was indicated as *P < 0.05, **P < 0.01, and ***P < 0.001, Two-way ANOVA test and Tukey's multiple-group comparison analysis vs. the control group and †P < 0.05 when comparing with the CCAO group.

Fig. 4

Combined prophylactic zinc and therapeutic selenium administration decreased astrogliosis in the hippocampus. The representative micrographs are integrated from several 20X amplifications of immunohistochemical staining. The optical density (OD) of GFAP is expressed on the graph; the background was subtracted from the measurement. The graph shows values expressed by the mean ± SEM of 24 slices per brain of 3 independent brains (Fig. 4E). The two-way ANOVA test with Bonferroni's correction was applied for multiple-group comparison against the control (*P < 0.05) and when compared with CCAO group (†P < 0.05).

Fig. 5

Prophylactic zinc and therapeutic selenium administrations decrease astrogliosis in the temporoparietal cortex. The representative micrographs are integrated from several 20X amplifications of immunohistochemical staining. The optical density (O.D.) of GFAP is expressed on the graph; the background was subtracted from the measurement. Values are the mean ± SEM of 24 slices of 3 independent brains. A two-way ANOVA test with Bonferroni's multiple-group comparison analysis. *P < 0.05 compared with the Control group and †P < 0.05 compared with the CCAO group.

Fig. 6

Prophylactic zinc and therapeutic selenium administrations prevented lipid peroxidation caused by ischemia in the temporoparietal cortex on day 34 post-reperfusion. Values represent the mean ± SEM of n = 6 rats. Statistical differences are indicated as *P < 0.05, two-way ANOVA with Dunnett's post hoc compared to the control group and †P < 0.05 with the CCAO group.

Fig. 7

Prophylactic administration of zinc and therapeutic administration of selenium increased BrdU-positive cells in the dentate gyrus of the hippocampus. The representative micrographs presented in the upper right corner of each micrograph were integrated from several 20X amplifications; the large photo corresponds to a magnification of the area marked with an arrow in the integrated micrograph. The number of BrdU-positive cells is expressed on the graph. Values are the mean ± SEM of 24 slices of 3 independent brains obtained each 240 μm. Statistical differences are indicated as P < 0.05, as determined by the Two-way ANOVA test and Bonferroni's multiple comparison analysis, compared to *, the control group, or †, the CCAO group. The arrows indicate BrdU immunoreactivity.

Fig. 8

Prophylactic administration of zinc and therapeutic administration of selenium increased BrdU-positive cells in the temporoparietal cortex. The graph shows the number of BrdU-positive cells in the control and experimental groups. Values are the mean ± SEM of 24 slices of 3 independent brains obtained each 240 μm. Statistical differences are indicated as P < 0.05, as determined by the Two-way ANOVA test and Bonferroni's multiple comparison analysis when compared to either the * Control group or † the CCAO group. Arrows indicate BrdU immunoreactivity.