Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Abstract

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investigated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal transient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining calbindin D28k immunoreactivity.

Keywords: calcium binding protein; hippocampus; ischemic tolerance; nerve regeneration; neural regeneration; neuroprotection; pyramidal neurons; transient cerebral ischemia.

Figure 1

Cresyl violet (CV) staining of the gerbil hippocampus in the sham (A, a), IPC + sham (B, b), ischemia (C, c, E, e, G, g, I, i, K, and k), and IPC+ ischemia (D, d, F, f, H, h, J, j, L, and l) groups. In the ischemia group, a few CV-positive cells were found in the stratum pyramidale (SP, asterisks) of the CA1 area from 4 days post-ischemia. However, CV-positive CA1 pyramidal cells in the IPC + ischemia group were well preserved after ischemia/reperfusion. CA: Cornu ammonis; DG: dentate gyrus; SO: stratum oriens; SR: stratum radiatum; IPC: ischemia preconditioning. Scale bars: 200 μm for A–L (low magnification photos); 60 μm for a–l (high magnification photos). (M) Relative analysis as percent in the mean number of CV-positive cells in the SP of the CA1 area (n = 7 in each group; *P < 0.05, vs. sham group; #P < 0.05, vs. corresponding ischemia group). The bars indicate the mean ± SEM.

Figure 2

Fluoro-Jade B (F-J B) histofluorescence staining of the CA1 area in the sham (A), IPC + sham (C), ischemia (B, E, F, I, and J), and IPC + ischemia (D, G, H, K, and L) groups. Many F-J B-positive cells were observed in the stratum pyramidale (SP, asterisks) 4 and 5 days (d) after ischemia/reperfusion; however, only a few F-J B-positive cells were observed in the IPC + ischemia group. SO: Stratum oriens; SR: stratum radiatum; IPC: ischemia preconditioning. Scale bar: 50 μm. (M) Relative analysis as percent in the mean number of F-J B-positive cells in the pyramidal layer of the CA1 area (n = 7 in each group; #P < 0.05, vs. ischemia group). The bars indicate the mean ± SEM.

Figure 3

Low magnification of calbindin-D28k (CB) immunohistochemistry in the hippocampus of the sham (A), IPC + sham (C), ischemia (B, E, F, I, and J), and IPC + ischemia (D, G, H, K, and L) groups. CB immunoreactivity was easily detected in the stratum pyramidale (SP) of the sham group; however, CB immunoreactivity was gradually decreased in the SP (asterisks) from 2 days (d) post-ischemia. CB-D28k immunoreactivity in the SP was well preserved in the IPC + sham and IPC + ischemia groups. Scale bar: 200 μm valid for A–L. High magnification of CB immunohistochemistry in the CA1 area of the sham (a), IPC + sham (c), ischemia (b, e, f, i, and j) and IPC + ischemia (d, g, h, k, and l) groups. CB immunoreactivity was hardly detected in the SP (asterisks) from 3 d post-ischemia. CB immunoreactivity in the SP of the IPC + ischemia group was well preserved after ischemia/reperfusion. SO: Stratum oriens; SR: stratum radiatum; IPC: ischemia preconditioning. Scale bar: 50 μm valid for a–l. (m) Relative immunoreactivity (RI) as percent values of CB immunoreactivity in the SP (n = 7 in each group; *P < 0.05, vs. sham group; #P < 0.05, vs. corresponding ischemia group; †P < 0.05, vs. respective pre-time point group). The bars indicate the mean ± SEM.