Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

Abstract

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min. 2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region. 3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group. 4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare. 5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.

Fig. 1.

CA1 region of hippocampus, immunohistochemical reaction with NeuN antibody. (A) NeuN positive pyramidal cells in CA1 region and granular cells in gyrus dentatus (GD) in the control section. (B) 20 min of ischemia/7 days of reperfusion induces neurodegeneration of CA1 pyramidal cells with no NeuN immunolabeling in contrast to NeuN positive CA2 pyramidal cells and granular cells of gyrus dentatus. Scale bar = 200 μm.

Fig. 1.

CA1 region of hippocampus, immunohistochemical reaction with NeuN antibody. (A) NeuN positive pyramidal cells in CA1 region and granular cells in gyrus dentatus (GD) in the control section. (B) 20 min of ischemia/7 days of reperfusion induces neurodegeneration of CA1 pyramidal cells with no NeuN immunolabeling in contrast to NeuN positive CA2 pyramidal cells and granular cells of gyrus dentatus. Scale bar = 200 μm.

Fig. 2.

Detail of CA1 region of hippocampus after NeuN immunohistochemical reaction. (A) NeuN immunoreactivity both in nuclei and cytoplasm in the control section. (B) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion shows intense NeuN immunoreaction in the nuclei and lower intensity of NeuN reaction in the shrinked CA1 pyramidal cells (^*). (C) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion with administration of EGb 761 shows decreased number of NeuN positive CA1 pyramidal cells with fiber-like material in the nucleus and immunolabeled cytoplasm (^*). Scale bar = 50 μm.

Fig. 2.

Detail of CA1 region of hippocampus after NeuN immunohistochemical reaction. (A) NeuN immunoreactivity both in nuclei and cytoplasm in the control section. (B) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion shows intense NeuN immunoreaction in the nuclei and lower intensity of NeuN reaction in the shrinked CA1 pyramidal cells (^*). (C) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion with administration of EGb 761 shows decreased number of NeuN positive CA1 pyramidal cells with fiber-like material in the nucleus and immunolabeled cytoplasm (^*). Scale bar = 50 μm.

Fig. 2.

Detail of CA1 region of hippocampus after NeuN immunohistochemical reaction. (A) NeuN immunoreactivity both in nuclei and cytoplasm in the control section. (B) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion shows intense NeuN immunoreaction in the nuclei and lower intensity of NeuN reaction in the shrinked CA1 pyramidal cells (^*). (C) Prophylactic treatment with EGb 761 for 7 days/20 min of ischemia/7 days of reperfusion with administration of EGb 761 shows decreased number of NeuN positive CA1 pyramidal cells with fiber-like material in the nucleus and immunolabeled cytoplasm (^*). Scale bar = 50 μm.

Fig. 3.

Detail of CA1 region, stratum oriens (SO), and stratum radiatum (SR) after GFAP immunoreaction. (A) GFAP-labeled astrocytes with thin processes in the control section (^*). (B) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion. (C) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion and EGb 761 administration. Scale bar = 50 μm.

Fig. 3.

Detail of CA1 region, stratum oriens (SO), and stratum radiatum (SR) after GFAP immunoreaction. (A) GFAP-labeled astrocytes with thin processes in the control section (^*). (B) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion. (C) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion and EGb 761 administration. Scale bar = 50 μm.

Fig. 3.

Detail of CA1 region, stratum oriens (SO), and stratum radiatum (SR) after GFAP immunoreaction. (A) GFAP-labeled astrocytes with thin processes in the control section (^*). (B) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion. (C) Reactive astrocytes (^*) in the experimental group with 7 days of EGb treatment/20 min of ischemia/7 days of reperfusion and EGb 761 administration. Scale bar = 50 μm.

Fig. 4.

Effect of EGb 761 (Tanakan=Tan) on hippocampal CA1 pyramidal cells after 20 min of ischemia/7 days of reperfusion. Surviving neurons immunolabeled with NeuN antibody were counted per mm in the middle of linear part of CA1. Results are presented as mean±SEM from five rats ^* p < 0.05.