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. 2006 Jun 7;26(7-8):1191–1202. doi: 10.1007/s10571-006-9080-6

Mapping of Rat Hippocampal Neurons With NeuN After Ischemia/Reperfusion and Ginkgo Biloba Extract (EGb 761) Pretreatment

Iveta Domoráková 1,3,✉, Jozef Burda 2, Eva Mechírová 1, Marianna Feriková 1
PMCID: PMC11520701  PMID: 16758319

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.

KEY WORDS: brain, hippocampus, CA1, ischemia, Ginkgo biloba extract (EGb 761), NeuN, GFAP, rat

INTRODUCTION

Ginkgo biloba is a herb often used as an alternative treatment for enhancing cognitive and other brain functions in healthy, as well as impaired populations. A standardized extract of the leaves of Ginkgo biloba (EGb 761) has neuroprotective effects (Seif-El-Nasr and El-Fattah, 1995; Gdovinová, 2001; Peng et al., 2003). Oral administration of EGb 761 has been shown to protect against neuronal death in the gerbil hippocampus after transient global ischemia (Spinnewyn et al., 1986; Chandrasekaran et al., 2001) and after focal ischemia (Zhang et al., 2000; Clark et al., 2001). The extract EGb 761 contains 24% flavonol glycosides, 6% terpene lactones (ginkgolides A, B, C, J and bilobalides (Drieu, 1986; DeFeudis, 1998). EGb 761 eliminates free oxygen radicals (Oyama et al., 1996) and reduces lipid peroxidation (Joyeux et al., 1995). It is generally hypothesized that neuro- and cardioprotection is associated with the free-radical scavenging and/or antioxidant properties of EGb 761. Extract of Ginkgo biloba has the ability to scavange free radicals (Seif-El-Nasr and El-Fattah, 1995; Westman et al., 2000) and bilobalides can inhibit production of reactive oxygen species in perfused heart tissue (Pietri et al., 1997). It was reported that EGb 761 could diminish the coagulation of platelets to inhibit thrombosis by antagonizing platelet activating factor (Kim et al., 1998). In the cardiovascular system, ginkgo extract can stimulate vasodilation that leads to increased blood flow and decreased blood pressure (Shen and Zhou, 1995).

Global brain ischemia in rodents causes delayed cell death in hippocampal CA1 pyramidal neurons several days after reperfusion. Other neurons such as hippocampal CA3 neurons are less vulnerable (Kirino, 1982; Pulsinelli, 1985). Similarly, delayed neuronal death was described in human hippocampus after cardiorespiratory arrest (Petito et al., 1987). If the ischemic period is transient and short, neuronal damage occurres only in selectively vulnerable areas (Pulsinelli et al., 1982a,b). In these regions the neurons are irreversibly damaged whereas glia and vascular cells survive (Petito and Babiak, 1982; Chen and Swanson, 2003). Astrocytes were previously considered as static cells that serve for neuronal support. Over the last decade a number of studies support the idea that astrocytes play an important role in the pathophysiology of cerebral ischemia. Recent studies showed that astrocytes are capable of producing trophic factors in ischemia—reperfusion injury (D'Ambrosio et al., 2001). Astrocytes maintain extracellular ionic environment (Leis et al., 2005) and are involved in metabolism of glutamate (Pascual et al., 1998; Matthias et al., 2003). Besides their metabolic role, astrocytes are considered to provide the major antioxidant defense of the brain (Swanson et al., 2004).

The present study investigates the protective effect of Ginkgo biloba extract against ischemia—reperfusion induced neuronal death produced by four-vessel occlusion protocol. NeuN immunohistochemistry was used for qualitative and quantitative expression of surviving neurons in the most vulnerable CA1 region in the rat hippocampus, as well as the response of astrocytes in the studied area.

METHODS

Handling of experimental animals was performed under the supervision of the Ethical Committee of Medical Faculty of P. J. Šafárik University in Košice.

Induction of Brain Ischemia and Treatment

The experiments were carried out in adult male Wistar rats (breeding colony of Medical Faculty of P. J. Šafárik University), weighing 280–300 g. Four groups of rats were kept in separate cages in standard laboratory conditions. Tanakan (Ginkgo biloba extract—EGb 761, IPSEN, France) was administered in the dose 40 mg/kg/day orally for 7 days. The animals were divided into four groups and each group included five animals.

  1. Control group (n=5).

  2. Experimental group with 20 min of ischemia and 7 days of reperfusion.

  3. Experimental group pretreated with Tanakan for 7 days, following 20 min of ischemia and 7 days of reperfusion without Tanakan administration.

  4. Experimental group pretreated 7 days with Tanakan, following 20 min of ischemia and 7 days of reperfusion with Tanakan administration.

Forebrain ischemia was induced by four-vessel occlusion model (Pulsinelli and Brierley, 1979) modified by Schmidt-Kastner et al. (1989). On the first day rats were anesthetized with 2.5% halothane and vertebral arteries were electrocauterized. On the following day, both common carotid arteries were occluded for 20 min by atraumatic clips under anesthesia induced with 2.5% halothane. Two minutes before carotid arteries occlusion, halothane was removed. During surgical intervention the normothermic conditions (37°C) were maintained. The severity of ischemia was evaluated by neurological examination. Criteria for forebrain ischemia were: loss of the righting reflex, mydriasis, paw extension. The rats that became unresponsive and lost the righting reflex during bilateral carotid artery occlusion and that showed no seizures during and after ischemia were used for the experiment. Only such animals are considered to have met the criteria for adequate ischemia (Pulsinelli et al., 1982a,b).

Following the reperfusion period the animals were deeply anesthetized with 10% chloralhydrate intraperitoneally (300 mg/kg) and were transcardially perfused with 0.9 saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Dissected brains were placed in the same fixative for 7 days and embedded in paraffin.

Immunohistochemistry

Immunohistochemistry was performed on 10 μm thick hippocampal coronal sections. Deparaffinized sections were preincubated with 2% normal horse serum (NHS) and 0.20% Triton X-100 in 0.1 M PBS for 1 h at room temperature (RT). Primary mouse monoclonal NeuN antibody (Chemicon) at a dilution of 1:500 in 2% NHS and 0.20% Triton X-100 and 0.1 M PBS (pH 7.4) was than applied overnight at 4°C. After washing in PBS (3×5 min), sections were incubated with a biotinylated secondary horse-anti-mouse IgG antibody (1:400, Vector Laboratories) for 2 h in dark at RT. After further washing in PBS, ABC Vecta stain kit (Vector Laboratories) was applied for 1 h in dark at RT. For detection of astrocytes sections were preincubated in 5% normal goat serum, 1% bovine serum albumin in 0.1 M PBS (pH 7.4) for 2 h following incubation in primary rabbit polyclonal anti-GFAP (1:80; Sigma) at 4°C overnight. Sections were rinsed in PBS (3×5 min), incubated in anti-rabbit biotinylated secondary antibody (1:20) for 2 h and then in extravidin-peroxidase (1:20) for 1 h (Rabbit ExtrAvidin Peroxidase Staining Kit, Sigma). For the peroxidase immunohistochemistry, both NeuN and GFAP immunoreaction was visualized using 3,3′-diaminobenzidine (DAB, Fluka). The sections were counterstained with Mayer's hematoxylin.

Image and Quantitative Analysis

The sections were examined by light microscope OLYMPUS and performed by OLYMPUS DP-SOFT.

Quantitative assessment of NeuN immunoreactivity in the hippocampal sections was performed by using Image tool software (UTHSCSA, San Antonio, USA). The number of NeuN immunoreactive pyramidal cells were counted per 1 mm of linear length in CA1 region.

Statistically significant differences between control and experimental samples were assessed by means of ANOVA followed by Tukey–Kramer's test. Statistical significance was defined as a value p < 0.05.

RESULTS

NeuN antibody labels nuclei and cytoplasm of most neuronal cell types in all regions of the adult brain including cerebral cortex, hippocampus, cerebellum, etc. No immunoreactivity was observed in astrocytes.

In control hippocampal sections NeuN immunoreactivity was seen in the pyramidal cells of CA1-4 subfields both in the nuclei and cytoplasm. The most cytoplasmic immunopositivity was concentrated in the soma, rarely it extended at a short distance into the processes. In granular cells of dentate gyrus expression of nuclear immunoreactivity to NeuN was more intense. Some interneurons of the hilar region were also immunoreactive (Figs. 1(A) and 2(A)). GFAP immunolabeling showed astrocytes with radially arranged thin processes. Astrocytes were dispersed in all layers of the hippocampus Fig. 3(A).

Fig. 1.

Fig. 1.

Fig. 1.

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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.

Fig. 2.

Fig. 2.

Fig. 2.

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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.

Fig. 3.

Fig. 3.

Fig. 3.

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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.

In the group of rats subjected to 20 min of ischemia and 7 days of reperfusion significant changes in NeuN immunoreactivity were found in the most vulnerable pyramidal cells of CA1 region. In the layer of CA1 no NeuN labeling was performed in contrast with CA2 NeuN positive pyramidal cells (Fig. 1(B)). In CA2-4 pyramidal cells and dentate gyrus granular cells no remarkable changes of NeuN immunopositivity were seen. Slightly reactive astrocytes were found in all hippocampal layers.

Protective effect of Tanakan was found in the group of rats with prophylactic treatment with Tanakan 7 days before 20 min of ischemia, following 7 days of reperfusion without Tanakan. Significant immunohistochemical changes were shown in the selectively vulnerable CA1 region. Well-expressed NeuN antibody was seen in CA1 pyramidal cells. Despite a mild shrinkage of their soma the cells showed NeuN positive cytoplasm, as well as an intense immunoreaction in the nuclei (Fig. 2(B)). The number of surviving neurons counted in the linear part of the CA1 region was 100.33±7.82 cells/mm (Fig. 4). In this experimental group a remarkable increase of GFAP expression was found in astrocytes predominantly in stratum oriens, stratum radiatum, and lacunosum (Fig. 3(B)).

Fig. 4.

Fig. 4.

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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.

In contrast to the findings of previously described experimental group, surprising results were obtained in the group of rats with 7 days of Tanakan pretreatment following 20 min of ischemia and 7 days of reperfusion with Tanakan administration. In hippocampal CA1 region decreased number of NeuN-immunolabeled pyramidal cells was found together with different cell morphology. The shape of these cells and intensity of cytoplasmic NeuN immunoreaction was the same as in the control section (Fig. 2(C)). Nuclear NeuN immunoreaction showed a fine network-like appearance. The number of surviving NeuN positive cells counted in linear length of CA1 was 59.5±2.74 cells/mm (Fig. 4). Pyramidal cells of CA2-4 and also granular cells of dentate gyrus were NeuN positive without noticeable differences. Increased number of reactive GFAP positive astrocytes (Fig. 3(C)) with thick processes were found predominantly in stratum oriens, lower part of stratum radiatum, and lacunosum.

DISCUSSION

The protective effects of Ginkgo biloba extract (EGb 761) to central nervous system has been previously demonstrated (Mechírová and Domoráková, 2002; Feriková et al., 2004). Oral administration of EGb 761 reduces neuronal death in gerbil hippocampus after transient global ischemia (Spinnewyn et al., 1986; Calapai et al., 2000), and protects against neuronal death in rats after focal ischemia induced by middle cerebral artery occlusion (Zhang et al., 2000).

Our study has shown neuroprotective effects of Ginkgo biloba extract on the most vulnerable population of CA1 pyramidal cells after 20 min of ischemia and 7 days of reperfusion. It was concluded that prophylactic administration of EGb 761 seven days before ischemia had beneficial effect for surviving neurons that are destined to die. Surprisingly, in another experimental group, administration of EGb 761 seven days before and 7 days after ischemia, has not caused expected beneficial effect. According to Peng et al. (2003), the effectiveness of prophylactic administration of EGb 761 was more satisfactory than postischemic administration. The reason of less neuronal survival in the animal group with pre- and posttreatment with EGb 761 than those in the group with only pretreatment can be explained by ischemia-induced tolerance. Some drugs, like free radical scavengers and antioxidants used shortly before ischemia or in the time of ischemia prevent acquisition of ischemic tolerance by negative influence on activation of endogenous protective mechanisms against ischemia (Puisieux et al., 2004).

It is well known that a chain reaction occurres in the brain after acute cerebral ischemia. The biochemical metabolic changes begin as the first ones. First minutes of postischemic reperfusion are characterized by nearly complete block of translation, focused mainly on the inhibition of initiation, which is manifested by disaggregation of polyribosomes (Bodsch et al., 1985; Burda et al., 1994). In the selectively vulnerable regions the inhibition of protein synthesis is persistent. Our previous study demonstrated that EGb 761 administration 7 days before 20 min of ischemia significantly improves protein synthesis rate in vitro in brain extracts (Hrehorovská et al., 2004).

The mechanisms of neuroprotection against ischemic injury by EGb 761 is not completely understood. The extract contains 24% flavonol glycosides and 3% bilobalides (DeFeudis, 1998) The flavonol glycosides have the ability to scavange free radicals and the extract is able to protect neurons against free radical injury (Marcocci et al., 1994; Seif-El-Nasr and El-Fattah, 1995). It was reported that mitochondria are the main site of free radical production in the cell (Fiskum et al., 1999). In normal conditions, reactive oxygen species (ROS) are eliminated by a very efficient antioxidant system constituted by enzymes, glutathione reductase, glutathione peroxidase, superoxide dismutase, vitamins C and E. Reactive oxygen species produced after ischemia—reperfusion are potentially very damaging for mitochondria (Dykens, 1994). The bilobalides, which contain flavonoids, organic acids, and terpenoids, have been reported to scavange hydroxyl, superoxide, peroxyl, and nitric oxide radicals and thus protect mitochondrial function (Roy et al., 1998; Morin et al., 2001).

In the present study it has been demostrated that reactive astrocytes could also affect neuronal survival after ischemia—reperfusion. Reactive astrocytes producing heat shock proteins, cytokines, and trophic factors have the effect on postischemic neuronal survival (Lo et al., 2003; Nishino and Nowak, 2004). Moreover, they are able to scavange ROS and remove extracellular glutamate (Swanson et al., 2004).

In conclusion, our results suggest that Ginkgo biloba extract (EGb 761) protects the most vulnerable CA1 pyramidal cells against ischemia—reperfusion in rats by inhibition of free oxygen radicals creation.

ACKNOWLEDGMENT

This study was supported by the grant VEGA 2/3219/23 and APVT 51-0219-04.

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