Elsevier

Neurobiology of Disease

Volume 24, Issue 2, November 2006, Pages 345-356
Neurobiology of Disease

Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion

https://doi.org/10.1016/j.nbd.2006.07.012Get rights and content

Abstract

To determine whether Bcl-2 could influence adult neurogenesis and prevent apoptosis of newborn neurons, we injected Bcl-2 expressing plasmid into the lateral ventricle of rat brain immediately following a 30-min occlusion of the middle cerebral artery (MCAO). We found that Bcl-2 increased neural progenitor cells (BrdU+-DCX+) in the ipsilateral striatum, newborn immature neurons (BrdU+-Tuj-1+) and newborn mature neurons (BrdU+-MAP-2+) in the ipsilateral striatum and frontal cortex at 1 to 4 weeks following MCAO. Bcl-2 overexpression promoted development of newborn neurons into GABAergic and cholinergic neurons in the ipsilateral striatum. Moreover, Bcl-2 significantly decreased the apoptosis of newborn neurons, determined by double staining of Tuj-1 and activated caspase-3 (Tuj-1+-Casp+). These results indicate that overexpression of Bcl-2 in adult rat brain enhances neurogenesis and survival of newborn neurons. Increasing neurogenesis and preventing the death of newborn neuron may be a strategy to aid in the repair of adult brain after stroke.

Introduction

Neurogenesis in the adult brain persists in at least two regions, the subgranular zone (SGZ) and the subventricular zone (SVZ) (Abrous et al., 2005). Although a low level of neurogenesis occurs in the healthy, uninjured brain, it is significantly enhanced following pathological events (Parent, 2003). Cerebral ischemia can stimulate proliferation and differentiation of neural progenitor cells in both the SGZ (Liu et al., 1998) and the SVZ (Zhang et al., 2001). Ischemia-induced neurogenesis is also seen in regions that are not a direct source of neurogenic precursors, including the hippocampal CA1 area (Nakatomi et al., 2002), cerebral cortex (Jiang et al., 2001) and striatum (Arvidsson et al., 2002, Nadareishvili and Hallenbeck, 2003), implying that newborn neurons can migrate toward damaged regions and replace injured neurons. Adult neurogenesis as a treatment to replace dying neurons in the injured brain is therefore a potentially significant treatment scheme for brain injury. Attention has been given to the study of factors that have the potential of enhancing neurogenesis in the adult brain, including growth factors, exercise and exposure to an enriched environment (Parent, 2003, Abrous et al., 2005). Unfortunately, the majority of newborn neurons in the adult brain do not persevere. Within 6 weeks of their birth, there is a 50% loss of newborn neurons in the adult olfactory bulb (Petreanu and Alvarez-Buylla, 2002) and dentate gyrus (Dayer et al., 2003). There is evidence that the loss of newborn neurons is caused by apoptosis. It is found a large number of cells positive for dUTP-nick end labeling (TUNEL) (Biebl et al., 2000) or caspase-3 (Bingham et al., 2005) in the same area containing bromodeoxyuridine (BrdU) immunoreactivity. Following ischemic stroke, 80% of newborn striatal neurons die from 2 to 6 weeks after the stroke event (Arvidsson et al., 2002). Although the mechanisms underlying apoptosis of newborn neurons are unclear, knockout of the pro-apoptotic bcl-2 family gene bax in mice increases the survival of newborn adult hippocampal neurons, suggesting that endogenous Bax participates in apoptosis of recently born adult neurons (Sun et al., 2004).

Bcl-2, an inhibitor of apoptosis in neuronal and glial cells, is highly expressed in the embryonic and developing mammalian brain (Abe-Dohmae et al., 1993, Ferrer et al., 1994). In the adult brain, however, it is restricted to progenitor rich-regions (Bernier and Parent, 1998, Bernier et al., 2000). Increasing evidence suggests that, in addition to its anti-apoptotic properties, Bcl-2 has an important function in cell differentiation and growth. Cultured sensory neurons isolated from the trigeminal ganglia of bcl-2-deficient mouse embryos extend axons more slowly than neurons from wild type mice (Hilton et al., 1997). On the other hand, cultured retinal ganglion neurons from bcl-2-overexpressing transgenic mice extend axons at a greater rate than the ganglion neurons from wild type mice (Chen et al., 1997). In vivo studies also indicate that Bcl-2 overexpression enhances retinal axon regeneration after optic-tract transaction (Chen et al., 1997) and increases axonal growth of transplanted fetal dopaminergic neurons in the rat striatum (Holm et al., 2001).

Augmenting neuronal replacement by enhancing the survival and maturation of endogenous progenitors is a potentially useful treatment following cerebral ischemia. The neuroprotective effects of Bcl-2 against ischemic brain injury are due to its anti-apoptotic effect; however, it is not known whether Bcl-2 has any influence on ischemia-induced neurogenesis. We therefore used MCAO model to induce transient focal brain ischemia. Following MCAO, neurogenesis was enhanced and apoptosis did occur in newborn neurons. In addition, the injection of Bcl-2 expressing plasmid into the lateral ventricle enhanced neurogenesis and the survival of newborn neurons in adult rat brains after MCAO.

Section snippets

Animals and reagents

Adult male Sprague–Dawley rats (220–250 g) from Shanghai Experimental Animal center, Chinese Academy of Sciences, were used in the study. The Medical Experimental Animal Administrative Committee of Shanghai approved all experiments. All efforts were made to minimize animal suffering and reduce the number of animals used.

Cresyl violet and diaminobenzidine (DAB) were purchased from Sigma (St. Louis, MO, USA). Fluoro-Jade B, guinea pig polyclonal anti-doublecortin (DCX), mouse monoclonal anti-βIII

Bcl-2 increased the proliferating neural progenitor cells in adult rat brain after MCAO

Bcl-2 has been detected in the SVZ of adult squirrel monkey and human brain (Bernier and Parent, 1998, Bernier et al., 2000). To determine any change of endogenous Bcl-2 expression in the adult rat brains after stroke, we performed Bcl-2 staining in brain sections of rats sacrificed at 7 days after a 30-min MCAO. We detected high density of Bcl-2-positive cells in the striatum surrounding the ischemic core as well as in the SVZ extensional regions (Figs. 1I and M), where is known to contain

Discussion

In the present study, we provide the first evidence that Bcl-2 overexpression by gene transfer enhances activation of neural progenitor cells, differentiation of newborn neurons into GABAergic and cholinergic neurons and reduces apoptosis of newborn neurons in rat brains following a transient occlusion of the middle cerebral artery. These findings suggest that Bcl-2 treatment could be beneficial to enhance neurogenesis and survival of newborn neurons in adult brain, which may be a strategy to

Acknowledgments

This work was supported by grants from the key Project of Chinese Ministry of Education (No. 2003-24), National Basic Research Program of China (No. 2006CB504100) and Shanghai Metropolitan Fund for Research and Development (02JC4025 and 04D214005) to Feng-Yan Sun.

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