Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

https://doi.org/10.1016/j.neures.2008.01.008Get rights and content

Abstract

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5′-Bromo-2′-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ).

Introduction

Stroke is the third most common cause of death in developed countries, exceeded only by coronary heart disease and cancer; also, represents the first cause of permanent disability above the age of 65 years. Despite that a restorative treatment has not been reached yet.

The adult central nervous system contains stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes (Emsley et al., 2005). It has been suggested that increased neurogenesis originating from stem cells located in subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) of adult brain in a variety of mammals, including humans, may promote morphological and functional recovery after cerebral ischemia, traumatic and primary degenerative brain diseases (Gould et al., 1999, Schmidt and Reymann, 2002).

It is known that transient global cerebral ischemia leads to delayed cell death of CA1 pyramidal neurons of the hippocampus in adult gerbils (Kirino, 1982, Schmidt-Kastner and Freund, 1991, Hara et al., 1993, Ferrer et al., 1997, Horiguchi et al., 2002), and that brain ischemia increases neurogenesis in the DG of the hippocampus of rodents including gerbils (Takagi et al., 1999, Sharp et al., 2002, Kokaia and Lindvall, 2003).

The origin of neural stem cells that give rise to new neurons in the adult CNS has been a subject of debate. Although neural progenitor proliferation and neurogenesis occur in SVZ and SGZ, several studies have demonstrated the production of new neurons in many other regions of the adult mammalian brain including CA1 area of gerbils after global cerebral ischemia (Schmidt and Reymann, 2002).

This and other issues like the mechanisms that trigger and regulate the proliferation and differentiation of neuronal progenitors are important to understand adult neurogenesis. Given the potential clinical applications and the limited understanding of this process, we decided to investigate the proliferation of progenitor cells and neurogenesis in the dentate gyrus and CA1 area of hippocampus at different time points after transient global cerebral ischemia by labelling dividing cells with the synthetic thymidine analogue BrdU, which is incorporated into the DNA of dividing cells in the S phase of the cell cycle and can be detected immunohistochemically (Gratzner, 1982, Kuhn et al., 1996). The expression of the glial marker Glial Fibrillary Acidic Protein (GFAP) was also studied. To determine the survival and fate of proliferating cells in the granule cell layer (GCL) of the DG and CA1 newborn cells were examined 4 weeks after BrdU administration using confocal microscopy.

Transient global cerebral ischemia was induced by occlusion of both common carotid arteries (CCA) for 5 min. In the gerbil brain, the lack of posterior communicating arteries between the carotid arteries and the vertebral arteries constitutes an incomplete Circle of Willis. Due to this unique anatomical structure, the gerbil has been widely used as an animal model to produce transient global cerebral ischemia following occlusion of both CCA (Ginsberg and Busto, 1989). However, because of the presence of substantial genetic heterogeneity in communicating arteries in gerbils, some animals have partial communicating vessels between the basilar and carotid circulations on one side (Wang et al., 2002). According to this and our results, hippocampal CA1 pyramidal neuronal death triggers proliferation in CA1 and neurogenesis in the DG, independently of the reduced blood flow.

Section snippets

Animal experimental design and BrdU injections

Procedures involving animals were in accordance with the guidelines established by the European Communities Council Directive of 24 November 1986 (86/609/EEC).

For proliferation studies, adult male mongolian gerbils (Meriones unguiculatus; 11–12 weeks of age; Harlan, Barcelona, Spain) weighing 60–65 g were divided into two groups: animals subjected to 5 min of global cerebral ischemia (n = 21) and sham operated animals (n = 15). Each group of gerbils was sacrificed (perfused transcardially) at three

Proliferation studies

Proliferation was defined as the number of newborn cells measured 24 h after a single injection of BrdU. Ischemic animals showed an increase in the number of proliferating cells in SGZ of the DG at 5 and 12 days after ischemia; these BrdU+ cells were not astrocytes because they did not express GFAP (Fig. 1). The number of BrdU+ cells in SGZ of the sham animals 5 days after surgery was 18.7 ± 2.5 (mean ± S.E.M.), this number increased to 34.6 ± 2.2 in the ischemic animals 5 days after ischemia, p < 0.05

Discussion

In the present study, we show that CA1 pyramidal cell layer death induces neurogenesis in dentate gyrus of gerbils and proliferative reaction in CA1 independently of the reduced cerebral blood flow.

One important issue with respect to the animal model used in this study is the presence of small vessels in the ventral surface of the midbrain that provide communication passages between the basilar and carotid circulations; this communication is sufficient to permit blood flowing into the carotid

Acknowledgment

This work was supported by Ministerio de Educación y Ciencia, Spain (SAF-2003-07207-C02-02, SAF2005-05086) and UTE project FIMA.

References (39)

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