Abstract

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

Highlights

  • The central nervous system (CNS) includes neural stem/progenitor cells (NSPCs) that are derived from neuroepithelial cells

  • According to reverse transcription polymerase chain reaction (RT-PCR), neurosphere-like cell clusters obtained from both areas showed NSPC markers, including Nestin and Sox2 as well as green fluorescent protein (GFP)

  • Consistent with the traits of CB-17 wild-type mice [10,11,17], the present results showed that ≥N6 Nestin-GFP mice (CB-17 background) had fewer arterial branches around the middle cerebral artery (MCA) compared with Nestin-GFP mice (C57BL/6 background)

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Summary

Introduction

The central nervous system (CNS) includes neural stem/progenitor cells (NSPCs) that are derived from neuroepithelial cells. Neuroepithelial cells produce radial glia, which later give rise to NSPCs localized in the subventricular zone (SVZ) of the adult brain [1] Under pathological conditions, such as after ischemic stroke, SVZ-derived NSPCs that express the NSPC marker Nestin are activated and migrate toward the ischemic areas [2]. Accumulating evidence shows that the migratory capacity of SVZ-derived NSPCs is limited [3,4] In addition to these conventional neurogenic zones, NSPCs are increasingly being shown to be regionally activated within and around the ischemic areas [5,6]. Consistent with these findings, we previously showed that, mature neural cells within the ischemic areas undergo cell death, Nestin+ NSPCs and/or

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