Keywords

1 Introduction

Vulnerability of the hippocampus to cerebral ischemia has been established in numerous studies. Selective and delayed neuronal cell death after global ischemia occurs in the subgranular zone (SGZ) in rats [1]. It was also reported that neurogenesis proceeds constantly in the subventricular zone (SVZ) and SGZ [2]. Neurogenesis in SVZ and SGZ is deeply involved in memory, learning and mood disorders. In rat SGZ, neurogenesis is enhanced by learning [3] and neuronal damage, such as seizure and ischemic insult, and reduced by post-ischemic stress [4]. Thus, the environment influences hippocampal cell death and neurogenesis due to cerebral ischemia. We previously reported that a stress environment before cerebral ischemia increases neuronal cell death and impairs neurogenesis [5]. Effects of an enriched environment (EE) have been reported in some studies of cerebral ischemia. For example, EE after ischemia increases neuronal cell death [6] and enhances neurogenesis in rat hippocampus [7]. Here, we evaluated the influence of EE prior to cerebral ischemia on post-ischemic neuronal cell death in rats.

2 Methods

All experiments were performed following an institutionally approved protocol in accordance with the Nihon University School of Medicine Guide for the Care and Use of Laboratory Animals.

Twenty-four Sprague-Dawley male rats (250–300 g) were anesthetized with isoflurane (1–1.2 %) in 30 % oxygen/70 % nitrous oxide. Their temperature was maintained at 37 °C with a heating pad. Femoral arteries were cannulated to monitor arterial blood pressure, pH and blood gases. Rats were assigned into the following groups: controls housed under standard conditions (SC) (n = 6); EE (housed in an EE without ischemia) (n = 6); SC + ischemia (n = 6); ischemia following EE (EE + ischemia) (n = 6). Global ischemia was induced by bilateral carotid arterial occlusion combined with hypotension for 10 min [8]. The EE consisted of a stainless steel cage (800 × 400 × 610 mm) containing wooden logs, tubes and shelves for climbing, and a running wheel. These rats were housed four per cage. On the other hand, in the SC group, rats were housed in pairs in standard laboratory cages (255 × 220 × 150 mm). All groups were housed SC or EE for 6 weeks.

Cognitive function was evaluated by means of the Y-maze test and a novel object recognition test (ORT) after 6 weeks SC or EE and after ischemia. Both tests have been used previously for cognitive assessment of rats [9–11]. Rats were euthanized at 7 days after ischemia. Reduction rates of Y-maze and ORT values were calculated as (pre-ischemia − post-ischemia)/pre-ischemia × 100 to assess differences between pre- and post-ischemia in the EE + ischemia group vs the corresponding differences in the SC + ischemia group. The brain was perfused with saline and 4 % paraformaldehyde, then 2 mm coronal sections were cut. Fluoro-Jade B staining and BrdU staining were used to evaluate hippocampal neuronal cell death and neurogenesis, respectively. Fluoro-Jade B-positive cells were counted in a 1 mm length of a horizontal section of the hippocampal CA1 area. BrdU-positive cells were counted in 1 mm2 of hippocampal dentate gyrus. Staining of phosphorylated cAMP response element-binding protein (phospho-CREB) was also performed in the two groups subjected to ischemia, and positive cells were counted in the same manner as in the case of Fluoro-Jade B staining. Positively stained cells in each case were evaluated in a double-blind manner. Data were expressed as mean ± SD. The significance of differences in positive cell counts was assessed by means of ANOVA followed by Tukey-Kramer tests. Differences with p ≤ 0.05 were considered significant.

3 Results

Hippocampal neuronal cell death in the two groups that had received global ischemia (SC + ischemia and EE + ischemia) was significantly increased compared with that in the two groups without ischemia (SC and EE) (Fig. 27.1a, b). Moreover, cell death in the EE + ischemia group was significantly decreased compared with that in the SC + ischemia group.

Fig. 27.1
figure 1figure 1

Evaluation of neuronal cell death by Fluoro-Jade B staining (a), (b). (a) Hippocampal neuronal cell death in the EE + ischemia group was significantly decreased compared with that in the SC + ischemia group. (b) The results of statistical analysis of cell counts of Fluoro-Jade B-stained cells. Evaluation of neurogenesis by BrdU staining (c), (d). (c) BrdU-positive cells in the SC + ischemia and EE + ischemia groups were significantly increased compared with those in the SC and EE groups. There was no significant difference between the SC + ischemia and EE + ischemia groups. (d) The results of statistical analysis of cell counts of BrdU-stained cells. Evaluation of neurogenesis by phospho-CREB staining (e), (f). (e) Phospho-CREB-positive cells in the SC + ischemia and EE + ischemia groups were significantly increased compared with those in the SC and EE groups. Phospho-CREB-positive cells were significantly increased in the EE + ischemia group compared with the SC + ischemia group. (f) The results of statistical analysis of cell counts of phospho-CREB-stained cells

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As for neurogenesis, BrdU-positive cells in the SC + ischemia and EE + ischemia groups were significantly increased compared with those in the SC and EE groups (Fig. 27.1c, d). However, no significant difference of BrdU-positive cells was observed between the SC + ischemia and EE + ischemia groups.

Phospho-CREB-positive cells in the SC + ischemia and EE + ischemia groups were also significantly increased compared with those in the SC and EE groups (Fig. 27.1e, f). Phospho-CREB-positive cells were significantly increased in EE + ischemia group compared with the SC + ischemia group.

Reduction rates in the two cognitive function tests in the EE + ischemia group were significantly lower than those in the SC + ischemia group (Fig. 27.2).In the case of ORT, there was no significant difference between before and after ischemia in the EE + ischemia group.

Fig. 27.2
figure 2figure 2

Plots a (Y-maze test) and c (novel object recognition test (ORT)) show the results of cognitive function tests of all four groups. Significant enhancement of cognitive function before ischemia was observed in the EE + ischemia group compared with the SC + ischemia group in both tests (p < 0.01). Plots b (Y-maze test) and d (ORT) show the reduction rates of cognitive function. The reduction rates in the two cognitive function tests in the EE + ischemia group were significantly lower than those in the SC + ischemia group

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4 Discussion

Exposure to EE after global cerebral ischemia leads to an increase in neuronal cell death and enhancement of neurogenesis in rat SGZ [6, 7]. However, little is known about whether EE prior to cerebral ischemia influences neuronal cell death and neurogenesis.

In the present study, exposure to EE prior to cerebral ischemia resulted in a decrease of neuronal cell death in the SGZ. This is a different response from that in rats exposed to EE after cerebral ischemia, because EE after cerebral ischemia increased neuronal cell death in rat hippocampus [6]. As regards neurogenesis, there was no difference between the EE + ischemia and SC + ischemia groups. The effect of EE prior to cerebral ischemia on neurogenesis may also be different from that of EE after ischemia. Expression of phospho-CREB in the SGZ was significantly increased in the EE + ischemia group compared with the SC + ischemia group. It was reported that the CREB pathway is activated by ischemic insult [12], and the CREB pathway is related to cognitive function and anti-apoptotic effect through COX-2 and Bcl-2, respectively [13]. Our results suggest that activation of the CREB pathway may be related to enhancement of cognitive function before and after ischemia, prevention of cognitive impairment associated with ischemia and reduction of neuronal cell death associated with EE prior to ischemia. Additionally, the reduction of neuronal cell death itself may lead to the prevention of cognitive impairment associated with ischemia in the EE + ischemia group.

In conclusion, EE before global cerebral ischemia may reduce hippocampal cell death, enhance cognitive function before and after ischemia and prevent cognitive impairment associated with ischemia, compared with controls. The CREB pathway may play an important role in the neuronal cell death and enhancement and protection of cognitive ability associated with EE.