Regulation of extracellular signal-regulated kinase 1/2 influences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

Abstract

Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor (U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and accelerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/reperfusion.

Keywords: Bax; DNA dependent protein kinase; apoptosis; brain injury; cerebral ischemia/reperfusion; extracellular signal-regulated kinase; hippocampus; nerve regeneration; neural regeneration.

Figure 1

Effects of extracellular signal-regulated kinase 1/2 pathway inhibition on the number of surviving neurons in the hippocampal CA1 region following cerebral ischemia in diabetic rats. (A–D) Hematoxylin-eosin stained sections of nerve cell morphological changes in hippocampal CA1 regions 48 hours after injury in the sham (NSO) group (A), normoglycemia global cerebral ischemia/reperfusion (NI/R) group (B), diabetic global cerebral ischemia/reperfusion (DCI) group (C) and DCI + U0126 group (D); arrows indicate dead cells (× 400). (E) The number of surviving neurons in the hippocampal CA1 region from each group. Data are expressed as mean ± SD from five rats at each time point, n = 40. Differences between the groups were compared using one-way analysis of variance and the Student-Newman-Keuls test. *P < 0.05, vs. NSO group; #P < 0.05, vs. NI/R group; †P < 0.05, vs. DCI group.

Figure 2

Effects of extracellular signal-regulated kinase 1/2 pathway inhibition on the neurological function of diabetic rats following cerebral ischemia. A lower score shows more serious functional damage. Data are expressed as mean ± SD from five rats at each time point. There were forty rats in each group. Differences between the groups were compared with one-way analysis of variance and the Student-Newman-Keuls test. *P < 0.05, vs. sham (NSO) group; #P < 0.05, vs. normoglycemia global cerebral ischemia-reperfusion (NI/R) group; †P < 0.05, vs. diabetic global cerebral ischemia-reperfusion (DCI) group.

Figure 3

Expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) in diabetic rats following cerebral ischemia (absorbance ratio of phosphorylated ERK1/2/β-actin). (A–D) Immunohistochemistry of phosphorylated ERK1/2 positive cells in CA1 region 24 hours after injury in sham (NSO), normoglycemia global cerebral ischemia/reperfusion (NI/R), diabetic global cerebral ischemia/reperfusion (DCI) and DCI + U0126 groups, respectively (arrows refer to positive cells, × 400). (E) Western blot photomicrographs of phosphorylated ERK1/2 protein expression in hippocampal CA1 region in the NSO, NI/R, DCI and DCI + U0126 groups, respectively. (F) ERK1/2 phosphorylation from each group was detected using western blot analysis. Data are expressed as mean ± SD from five rats at each time point, 40 rats per group. Differences between the groups were compared using one-way analysis of variance and the Student-Newman-Keuls test. *P < 0.05, vs. NSO group; #P < 0.05, vs. NI/R group; †P < 0.05, vs. DCI group. h: Hours.

Figure 4

Effect of extracellular signal-regulated kinase 1/2 pathway inhibition on Ku70 activity in the hippocampal CA1 region following cerebral ischemia in diabetic rats (absorbance ratio of Ku70/β-actin). (A–D) Immunohistochemistry of Ku70 positive cells in hippocampal CA1 region 24 hours after injury in the sham (NSO), normoglycemia global cerebral ischemia/reperfusion (NI/R), diabetic global cerebral ischemia/reperfusion (DCI), and DCI + U0126 groups, respectively (arrows refer to positive cells, × 400). (E) Western blot photomicrographs of Ku70 protein expression in hippocampal CA1 regions in the NSO group, NI/R group, DCI group and DCI + U0126 group. (F) Ku70 expressions from each group were detected by western blot analysis. Data are expressed as mean ± SD from five rats at each time point, 40 rats per group. Differences between the groups were compared using one-way analysis of variance and the Student-Newman-Keuls test. *P < 0.05, vs. NSO group; #P < 0.05, vs. NI/R group; †P < 0.05, vs. DCI group. h: Hours.

Figure 5

Effect of extracellular signal-regulated kinase 1/2 pathway inhibition on Bax expression in the hippocampal CA1 region following cerebral ischemia in diabetic rats (absorbance ratio of Bax/β-actin). (A–D) Immunohistochemistry of Bax positive cells in hippocampal CA1 region 24 hours after injury in sham (NSO), normoglycemia global ce-rebral ischemia/reperfusion (NI/R), diabetic global cerebral ischemia/reperfusion (DCI), and DCI + U0126 groups, respectively (arrows refer to positive cells, × 400). (E) Western blot photomicrographs of Bax protein expression in hippocampal CA1 region in the NSO group, NI/R group, DCI group and DCI + U0126 group. (F) Bax expression from each group was detected by western blot analysis. Data are expressed as mean ± SD from five rats at each time point, 40 rats per group. Differences between the groups were compared using one-way analysis of variance and the Stu-dent-Newman-Keuls test. *P < 0.05, vs. NSO group; #P < 0.05, vs. NI/R group; †P < 0.05, vs. DCI group. h: Hour.