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WIREs Nanomed Nanobiotechnol
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In situ labeling and imaging of endogenous neural stem cell proliferation and migration

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Abstract Endogenous neural stem cells (eNSCs) reside in defined regions of the adult brain and have the potential to generate new brain cells, including neurons. Stimulation of adult neurogenesis presents an enormous potential for regenerative therapies in the central nervous system. However, the methods used to monitor the proliferation, migration, differentiation, and functional integration of eNSCs and their progeny are often invasive and limited in studying dynamic processes. To overcome this limitation, novel techniques and contrast mechanisms for in vivo imaging of neurogenesis have recently been developed and successfully applied. In vivo labeling of endogenous neuronal progenitor cells in situ with contrast agents or tracers enables longitudinal visualization of their proliferation and/or migration. Labeling of these cells with magnetic nanoparticles has proven to be very useful for tracking neuroblast migration with MRI. Alternatively, genetic labeling using reporter gene technology has been demonstrated for optical and MR imaging, leading to the development of powerful tools for in vivo optical imaging of neurogenesis. More recently, the iron storage protein ferritin has been used as an endogenously produced MRI contrast agent to monitor neuroblast migration. The use of specific promoters for neuronal progenitor cell imaging increases the specificity for visualizing neurogenesis. Further improvements of detection sensitivity and neurogenesis‐specific contrast are nevertheless required for each of these imaging techniques to further improve the already high utility of this toolbox for preclinical neurogenesis research. WIREs Nanomed Nanobiotechnol 2012, 4:663–679. doi: 10.1002/wnan.1192 This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease

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Reporter gene‐based neurogenesis imaging. (a) In vivo longitudinal follow‐up of stem cell migration with BLI at 2, 4, and 8 weeks postlabeling with a lentiviral vector expressing firefly luciferase and eGFP, injected at the SVZ. From 4 weeks post injection (p.i.), an additional focus was detected at the OB projection site, as well as the original focus at the site of injection (SVZ) (white arrows). (Reprinted with permission from Ref 52. Copyright 2008 John Wiley & sons) (b) BLI acquired in vivo from a 3‐month‐old doublecortin (Dcx) promoter‐luciferase transgenic mouse. Although BLI provides a rather low morphological resolution, the contour of structures bearing most neuronal precursors (subventricular regions and OB) can be distinguished based on their higher signal intensities. (c) Axial projection from several contiguous slices from T2*‐weighted in vivo dataset 10 days after chimeric ferritin‐encoding adenovirus injection in the SVZ. The arrow points to a hypointense trail of migrating neuroblasts along the RMS. Asterisks indicate the injection sites. There is no similar feature on the contralateral GFP control side. (Reprinted with permission from Ref 33. Copyright 2012 Elsevier) (d) Ex vivo MR image 30 weeks after injection of a ferritin‐encoding lentiviral vector in the right SVZ after bias field correction.64 The right OB shows overall diffuse hypointense contrast accumulation (white arrow) compared to the left OB, due to ferritin‐labeled neuroblasts arriving and integrating in the right OB.

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Adult rodent neurogenesis. Sagittal view on the rodent brain showing the two main sites of neurogenesis: the DG of the hippocampus and the SVZ of the lateral ventricle. Neural progenitor cells from the SVZ migrate through the RMS and differentiate into interneurons in the OB. The inset shows the structural organization of the SVZ. Slowly proliferating astroglial‐like type B cells generate actively proliferating type C cells, which in turn give rise to migrating neuroblasts or type A cells. These different cell populations can be distinguished on the basis of several immunohistochemical markers: type B cells are positive for GFAP, type C cells and type A cells are dcx‐positive, and mature interneurons are NeuN‐positive. Abbreviations: CC, corpus callosum; dcx, doublecortin; DG, dentate gyrus; E, ependymal cells; GFAP, glial fibrillary acidic protein; LV, lateral ventricle; NeuN, neuronal nuclear antigen; OB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone. (Reprinted with permission from Ref 52. Copyright 2008 John Wiley & Sons)

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In vivo [18F]FLT‐PET corresponds to sites of eNPC proliferation. (a) Adult rats injected with [18F]FLT showed elevated tracer binding in the SVZ in vivo ([18F]FLT‐PET matched on MRI‐atlas of rat brain; white box in upper image is magnified in a′ below). (b) BrdU accumulation in proliferating cells in the SVZ corresponded with the [18F]FLT signal (×10). Scale bars: 1 mm. (Reprinted with permission from Ref 95. Copyright 2010 Society of Neuroscience).

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Fluorescent reporter genes for neurogenesis imaging. (a) Immunohistology of eGFP‐positive neuroblasts 2 weeks after lentiviral vector‐mediated labeling at the SVZ. GFP‐labeled neuroblasts migrate from the SVZ along the RMS toward the OB. (b) Time course of GFP‐labeled neuroblasts arriving and integrating continuously in the different layers of the OB from 2 days to 2 months after labeling. EPl, external plexiform layer; Gl, glomerular layer; Gr, granular layer; SEL, subependymal layer. Scale bar: 100 µm. (Reprinted with permission from Ref 50. Copyright 2006 Mary Ann Liebert, Inc.) (c) Time resolved activation of fluorescent reporter gene (eGFP) using a Dcx promoter‐CreERT allows for fate analysis of labeled cells on histological tissue sections. In the dentate gyrus, cells detected 3 days after Dcx promoter‐CreERT driven reporter activation had neuronal precursor morphology (left panel). Many cells labeled at this early time point were located at the subgranular lining of the dentate gyrus and displayed a morphology corresponding to very early differentiation stages (arrow heads). In contrast, 30 days after activation (right panel), most labeled cells presented a typical granule cells morphology with a cell soma located within the granular layer (scale bar: 25 µm; blue: nuclear counterstain DAPI).

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MRI of micron‐sized iron oxide (MPIO)‐labeled eNPC migration to the OB. (a) Schematic sagittal overview of the SVZ–RMS–OB neuroblast migration pathway. Abbreviations: CC, corpus callosum; LV, lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone. (b) Ex vivo MRI of a mouse injected with MPIO particles in the lateral ventricle at 8 weeks post‐injection, showing hypointense contrast spots along the RMS and spread in the OB. (Reprinted with permission from Ref 21. Copyright 2010 Elsevier) (c) Sagittal projection from several contiguous slices from T2*‐weighted in vivo dataset 10 days after chimeric ferritin‐encoding adenovirus injection in the SVZ showing the injection site (asterisk). The arrow points to a hypointense trail of migrating neuroblasts along the RMS. (Reprinted with permission from Ref 33. Copyright 2012 Elsevier) (d) Perls' iron stain of brain sections from the same experiment as in (c) shows iron rich (blue) cells, indicating that cells in brain regions of chimeric ferritin transduction showing MRI contrast also display iron accumulation. High magnification views are shown on the panels to the right. Blue cells are also apparent along the white matter tracks (top), proximal to the OB (middle), and in the RMS (bottom). (Reprinted with permission from Ref 33. Copyright 2012 Elsevier) (e) Serial MR images (minimal intensity projections over 390 µm) of MPIO‐labeled neuroblasts 4, 6, and 12 weeks after injection in the lateral ventricle, showing hypointense contrast along the RMS and spreading out in the OB. (f–i) Immunohistochemistry and electron microscopy from a mouse injected with 10 µL MPIOs in the lateral ventricle. (f–h) Fluorescence images show the presence of green fluorescent MPIOs in the SVZ, the RMS, and the OB. Dcx+ (red‐stained) migrating NPCs labeled with MPIOs are present in the dorsal portion of the SVZ (f) and the RMS (g). (Reprinted with permission from Ref 21. Copyright 2010 Elsevier) A Dcx+ (red) and NeuN+ (blue) cell containing MPIOs (white arrow) in the OB is shown in (h); the double staining indicates that this NPC is differentiating into a mature neuron. (i) Electron microscopic image at the level of the SVZ shows iron containing vesicles (black arrow) located in the cytoplasm near the nucleus of an astrocyte‐like progenitor cell. (Reprinted with permission from Ref 21. Copyright 2010 Elsevier)

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