Elsevier

NeuroImage

Volume 48, Issue 2, 1 November 2009, Pages 319-328
NeuroImage

In vivo MRI of endogenous stem/progenitor cell migration from subventricular zone in normal and injured developing brains

https://doi.org/10.1016/j.neuroimage.2009.06.075Get rights and content

Abstract

Understanding the alterations of migratory activities of the endogenous neural stem/progenitor cells (NSPs) in injured developing brains is becoming increasingly imperative for curative reasons. In this study, 10-day-old neonatal rats with and without hypoxic–ischemic (HI) insult at postnatal day 7 were injected intraventricularly with micron-sized iron oxide particles (MPIOs), followed by serial high-resolution MRI at 7 T for 2 weeks. MRI findings were correlated to the histological analysis using iron staining and several immunohistochemical double staining. The results indicated that in normal and HI-injured brains the NSPs from the subventricular zone (SVZ) were labeled by MPIOs, and migrated as newly created cells (iron+/BrdU+), neuroblasts (iron+/nestin+), astrocytes or astrocytes-like progenitor cells (iron+/GFAP+), and mature neurons (iron+/NeuN+). In normal brains, the endogenous NSPs mainly exhibited a tangential pattern in both rostral and caudal directions. The NSP radial migratory pattern could be observed in some rats. In the HI-injured brains during the same developmental period, the NSPs mainly migrated towards the HI lesion sites. The tangential, rostrocaudal migrations could be observed but impaired. These findings suggest that the NSP migratory pathways in SVZ change in response to the HI insult, likely due to the self-repairing efforts known in the neonatal brains. The MRI approach demonstrated here is potentially applicable to the in vivo and longitudinal study of NSP cell activities in developing brains under normal and pathological conditions and in therapeutic interventions.

Introduction

Subventricular zone (SVZ) is a continual germinal zone surrounding the ventricles, which expands prominently during the latter third trimester of prenatal period and remains constant thereafter throughout the mammalian life. It is the largest source of neural stem cells and transit-amplifying progenitor cells that can generate progeny during mammalian forebrain development (Brazel et al., 2003, Marshall et al., 2003). Since currently it is not possible to clearly discern neural stem cells from the multi-potential progenitors that possess limited self-renewal especially in neonatal SVZ, the term neural stem/progenitor cells (NSPs) has been adopted to encompass both populations (Felling et al., 2006, Yang and Levison, 2006). In the postnatal brain, the NSPs that reside within the SVZ generate the olfactory interneurons and most of glia cells in the forebrain (Brazel et al., 2003, Marshall et al., 2003). While enormous progress has been made in recent decades in uncovering the contributions of NSPs in SVZ to normal brain development, it is becoming increasingly imperative to investigate the alterations of NSP behavior after brain injuries during early postnatal period. Perinatal hypoxia–ischemia (HI) is the leading cause of neurological disorders resulting from birth complications. Approximately 50% of survivors will develop neurological sequelae such as cerebral palsy, epilepsy, and cognitive deficits (Felling et al., 2006, Wood et al., 2000, Yang and Levison, 2006). Previous studies have demonstrated that the NSPs within SVZ provide a great potential to regenerate not only the neurons but also various glial cells required to reconstitute a fully functional developmental brain (Felling et al., 2006, Iwai et al., 2006, Ong et al., 2005, Romanko et al., 2004a, Yang et al., 2007, Yang and Levison, 2006, Yang and Levison, 2007). Thus the ability to identify and track NSPs in vivo could greatly help us understand the brain responses to injuries and design therapeutic interventions for exploration of their curative potential.

At present, electron microscopy, immunohistochemistry and fluorescence imaging are the standard techniques for identifying the migration of NSPs from SVZ. They are often detected dynamically by means of retroviruses (Suzuki and Goldman, 2003, Walsh and Cepko, 1988), thymidine (Altman and Das, 1966, Lois and Alvarez-Buylla, 1994) or bromodeoxyuridine (BrdU) labeling (Hayashi et al., 2005, Yang and Levison, 2006). Recently, the transgenic mice expressing green fluorescent protein (GFP) driven by nestin promoter and multiphoton microscopy for detecting nestin-GFP cells have been successfully demonstrated to study the NSPs in the rostral migratory stream (RMS) in both intact and ischemic brains in young mice (Zhao and Nam, 2007). Despite the numerous studies investigating the NSP migration from SVZ using ex vivo tissue processing, there is very limited knowledge regarding the in vivo NSP migration within the whole brain. Although positron emission tomography is an in vivo and non-invasive method for monitoring cell metabolism, it alone cannot provide sufficient spatial information to characterize the migrational activities (Cicchetti et al., 2007).

Magnetic resonance (MR) cellular imaging is an emerging technique for tracking the cell migration in vivo and non-invasively. It provides the opportunity to gain a sufficiently high spatial resolution, relate functional measures with presence of cells in target organ, and repeat analysis at multiple time points (Bulte and Kraitchman, 2004, Cicchetti et al., 2007, Hoehn et al., 2002, Hoehn et al., 2007, Modo et al., 2002). Superparamagnetic iron oxide nanoparticles are commonly used as the exogenous contrast agent for cell labeling, which can be endocytosed by a variety of cell types and produce a strong signal loss in T2⁎-weighted imaging (T2∗WI) by virtue of their susceptibility difference from the adjacent environment (Bulte and Kraitchman, 2004, Hoehn et al., 2007, Panizzo et al., 2009). Recently, Shapiro et al. demonstrated that the micron-sized iron oxide particles (MPIOs) have certain advantages for MRI cell tracking (Shapiro et al., 2004, 2005, 2006a,bShapiro et al., 2007, Sumner et al., 2009). These large and relatively stable MPIOs can induce substantial susceptibility effect so that each cell with a single MPIO can be detected by MRI (Shapiro et al., 2004, 2006b, 2007). Moreover, MPIOs can be impregnated with various fluorescent dyes, permitting further detection of labeled cells by ex vivo immunofluorescence analysis (Shapiro et al., 2004Shapiro et al., 2006a, Sumner et al., 2009).

There are three approaches to label cells for MRI tracking (Hoehn et al., 2007). The first is in vitro labeling of stem cells or tumor cells for their post-transplantation MRI visualization. Such cell labeling is a time-consuming process and maintaining stem cells in undifferentiated state in culture is sometimes difficult (Guzman et al., 2007, Hoehn et al., 2002, Modo et al., 2002, Shapiro et al., 2006a). The second approach is systemic administration of MRI contrast agent for macrophage imaging, which has been reported so far to observe inflammatory processes in stroke, experimental autoimmune encephalomyelitis, graft rejection and arthritis (Bulte and Kraitchman, 2004, Hoehn et al., 2007, Wiart et al., 2007, Wu et al., 2006). The third approach is in situ cell labeling for monitoring neurogenesis by direct injection of MPIOs or iron oxide nanoparticles into the ventricles near the SVZ. Even though the uptake of MPIOs or iron oxide nanoparticles by the precursors in the SVZ is small (∼ 30%) in adult rodents (Sumner et al., 2009), several MRI studies have successfully demonstrated the migration of labeled cells along the RMS into the olfactory bulb (OB) in normal adult rats (Panizzo et al., 2009, Shapiro et al., 2006a, Sumner et al., 2009). This approach offers the unique advantage of selective in situ labeling for study of NSPs in vivo.

It is known that the NSPs from the SVZ in the early postnatal brains are distinct from those in the adult brains in their migratory pathways (Brazel et al., 2003, Marshall et al., 2003). Few MRI studies published so far have exclusively focused on the NSP migration in normal and adult rat brains. There has been no report of in vivo MRI for tracking NSP activities in normal or injured developing brains. The objective of the present study was to employ the in situ cell labeling approach to characterize the migration of the endogenous NSPs from the SVZ in the postnatal developing rat brains in normal and HI-injured developing conditions. We further hypothesized that the NSP migrating patterns in normal and HI-injured developing brains would differ and could be detected by serial high-resolution in vivo MRI in addition to postmortem immunohistochemical analysis. Such in vivo MRI analysis can improve our understanding of NSP behavior in the developing mammalian brains and its alterations during injuries.

Section snippets

Animal preparation

Pregnant Sprague–Dawley rats were obtained approximately 2 days before parturition. Neonate rats were kept with their mother in regular light/dark cycles for 7 days after birth. Twelve postnatal day 7 (P7) rats (12–16 g) were divided into two groups, 6 in normal group and 6 in HI group. Neonatal rats in HI group underwent HI insult at P7. Briefly, unilateral ligation of left common carotid artery was performed in each pup under isoflurane anesthesia. The surgery usually lasted 5 min for each

MRI of migrating NSPs and histological colocalization in the normal developing brains

At 1 day post injection (dpi), the dark contrast was seen to permeate into both sides of lateral ventricle in the 3 neonatal rats in the normal group while the remaining 3 had the dark contrast mainly in the left lateral ventricle. Between 3 and 14 dpi, the dark contrast in lateral ventricle pervaded into the 3rd ventricle (3V) and the arachnoid space, such as the hippocampal fissure (HF) (black arrows in 1st and 2nd rows in Fig. 2) in all rats. A tangential migratory pattern was observed

Disscusion

Previous studies have shown that the SVZ neural stem/progenitor (NSP) cell migration pathways differ between neonatal and adult periods (Brazel et al., 2003, Doetsch and Alvarez-Buylla, 1996, Levison et al., 1993, Marshall et al., 2003, Suzuki and Goldman, 2003), and alter after ischemic injuries (Arvidsson et al., 2002, Brazel et al., 2004, Felling et al., 2006, Gotts and Chesselet, 2005b, Macas et al., 2006, Romanko et al., 2004a, Yang et al., 2007). However, the methods used largely relied

Conclusion

In this study, we demonstrated that intraventricular injection of MPIOs at P10 allows the MRI visualization of the migrations of neural stem/progenitor cells (NSPs) in subventricular zone (SVZ) in normal and injured developing rat brains. In normal brains, the endogenous NSPs mainly exhibited a tangential, rostrocaudal migration pattern from P10 to P24. The NSP radial migratory pattern could be observed but less clearly. For the hypoxia–ischemia (HI) injured postnatal brains during the same

Acknowledgments

This work was supported in part by research grants from the Hong Kong Research Grant Council (HKU 7793/08M) and the Chinese National Science Foundation Council (30770673).

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