Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

Abstract

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1-3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.

Keywords: CA1 region; cerebral ischemia/reperfusion; delayed neuronal death; immunohistochemistry; neural regeneration; p53 tumor suppressor gene family; pyramidal neurons; western blotting.

Figure 1

Neuronal nuclei (NeuN) immunohistochemistry and Fluoro-Jade B (F-J B) histofluorescence staining in the hippocampal CA1 region of the adult (left three columns) and young (right three columns) gerbils in the sham- (A–F) and ischemia-operated (G–R) groups. In the ischemia-operated adult groups, a few NeuN immunoreactive neurons (black arrows) and many F-J B positive cells (white asterisk) were shown in the stratum pyramidale of the hippocampal CA1 region 4 days after ischemia/reperfusion; however, at this time point, neuronal damage is hardly found in the ischemia-operated young group. Seven days after ischemia/reperfusion, decreased NeuN immunoreactive neurons (black asterisk) and increased F-J B positive cells (white arrows) were observed in the stratum pyramidale of the hippocampal CA1 region in the ischemia-operated young group. CA: Cornus ammonis; DG: dentate gyrus; SO: stratum oriens; SR: stratum radiatum. Scale bars: 800 μm in A, D, G, J, M, P, and 50 μm in B, C, E, F, H, I, K, L, N, O, Q, R.

Figure 2

Immunohistochemistry for p63 in the hippocampal CA1 region (left two columns) and CA2/3 regions (right two columns) of the adult and young gerbils in the sham- (A, B, a and b) and ischemia-operated (C–H and c–h) groups. In the sham-operated young group, p63 immunoreactivity in the stratum pyramidale (SP, black asterisks) was significantly lower than that in the sham-operated adult group. In the ischemia-operated adult group, p63 immunoreactivity was significantly decreased in the SP (black asterisk) of the hippocampal CA1 region 4 days after ischemia/ reperfusion. However, at this time point, p63 immunoreactivity was distinctively increased in the SP (white asterisk) of the ischemia-operated young group, and p63 immunoreactive pyramidal neurons were significantly decreased 7 days after ischemia/reperfusion. Meanwhile, in the ischemia-operated adult group, p63 immunoreactivity was not changed in the SP of the hippocampal CA2/3 regions compared with that in the sham-operated adult group. In the ischemia-operated young group, p63 immunoreactivity was similar to that in the ischemia-operated adult group. SO: Stratum oriens; SR: stratum radiatum. Scale bars: 50 μm in A–H and 100 μm in a–h.

Figure 3

Western blot analysis of p63 protein in the hippocampal CA1 region of the adult and young gerbils in the sham- and ischemia-operated groups. In the sham-operated young group, the protein level of p63 was significantly lower compared with that in the sham-operated adult group. Four days after ischemia/reperfusion, p63 protein level in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. Each experiment was repeated three times, and relative optical density (ROD) was represented as % values of immunoblot band. Data were analyzed by one-way analysis of variance (ANOVA) with a post hoc Bonferroni's multiple comparison test (*P < 0.05, vs. corresponding sham-operated group, #P < 0.05, vs. corresponding pre-time point group, †P < 0.05, vs. corresponding adult group). The bars indicate the means ± SEM. d: Day(s).