In Vivo Imaging of Dorsal Root Regeneration: Rapid Immobilization and Presynaptic Differentiation at the CNS/PNS Border

Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 03/2011; 31(12):4569-82. DOI: 10.1523/JNEUROSCI.4638-10.2011
Source: PubMed


Dorsal root (DR) axons regenerate in the PNS but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the CNS. Earlier studies that relied on conventional tracing techniques or postmortem analyses attributed the regeneration failure to growth inhibitors and lack of intrinsic growth potential. Here, we report the first in vivo imaging study of DR regeneration. Fluorescently labeled, large-diameter DR axons in thy1-YFPH mice elongated through a DR crush site, but not a transection site, and grew along the root at >1.5 mm/d with little variability. Surprisingly, they rarely turned around at the DREZ upon encountering astrocytes, but penetrated deeper into the CNS territory, where they rapidly stalled and then remained completely immobile or stable, even after conditioning lesions that enhanced growth along the root. Stalled axon tips and adjacent shafts were intensely immunolabeled with synapse markers. Ultrastructural analysis targeted to the DREZ enriched with recently arrived axons additionally revealed abundant axonal profiles exhibiting presynaptic features such as synaptic vesicles aggregated at active zones, but not postsynaptic features. These data suggest that axons are neither repelled nor continuously inhibited at the DREZ by growth-inhibitory molecules but are rapidly stabilized as they invade the CNS territory of the DREZ, forming presynaptic terminal endings on non-neuronal cells. Our work introduces a new experimental paradigm to the investigation of DR regeneration and may help to induce significant regeneration after spinal root injuries.

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    • "Proteoglycans produced by astrocytes in response to dorsal root injury are generally viewed as factors that prevent injured sensory axons from re-entering the adult spinal cord [26]. More recent studies have provided evidence that growing sensory axons are not repelled when they reach the DRTZ, but cease to grow after making synapse-like contacts with astrocytes within the spinal cord [27]. The GFAP-positive cells in the tubes thus appear to lack factors that induce stable contacts between growing sensory axons and bNCSC associated astrocytes. "
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    ABSTRACT: Background The boundary cap is a transient group of neural crest-derived cells located at the presumptive dorsal root transitional zone (DRTZ) when sensory axons enter the spinal cord during development. Later, these cells migrate to dorsal root ganglia and differentiate into subtypes of sensory neurons and glia. After birth when the DRTZ is established, sensory axons are no longer able to enter the spinal cord. Here we explored the fate of mouse boundary cap neural crest stem cells (bNCSCs) implanted to the injured DRTZ after dorsal root avulsion for their potential to assist sensory axon regeneration. Results Grafted cells showed extensive survival and differentiation after transplantation to the avulsed DRTZ. Transplanted cells located outside the spinal cord organized elongated tubes of Sox2/GFAP expressing cells closely associated with regenerating sensory axons or appeared as small clusters on the surface of the spinal cord. Other cells, migrating into the host spinal cord as single cells, differentiated to spinal cord neurons with different neurotransmitter characteristics, extensive fiber organization, and in some cases surrounded by glutamatergic terminal-like profiles. Conclusions These findings demonstrate that bNCSCs implanted at the site of dorsal root avulsion injury display remarkable differentiation plasticity inside the spinal cord and in the peripheral compartment where they organize tubes associated with regenerating sensory fibers. These properties offer a basis for exploring the ability of bNCSCs to assist regeneration of sensory axons into the spinal cord and replace lost neurons in the injured spinal cord.
    BMC Neuroscience 05/2014; 15(1):60. DOI:10.1186/1471-2202-15-60 · 2.67 Impact Factor
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    • "These observations are consistent with the hypothesis that regenerating axons form synapse-like terminals with reactive glia, which was originally advanced by Carlstedt (1985). Advances in imaging have allowed live in vivo studies of acute and chronically injured axons in the lesion environment (Di Maio et al., 2011; Evans et al., 2014; Farrar et al., 2012; Kerschensteiner et al., 2005; Ylera et al., 2009). Ylera et al. (2009) recently used in vivo imaging to demonstrate that chronically injured axons can, indeed, be aroused into a robust regenerative state. "
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    ABSTRACT: Astrocytes react to CNS injury by building a dense wall of filamentous processes around the lesion. Stromal cells quickly take up residence in the lesion core and synthesize connective tissue elements that contribute to fibrosis. Oligodendrocyte precursor cells proliferate within the lesion and help to entrap dystrophic axon tips. Here we review evidence that this aggregate scar acts as the major barrier to regeneration of axons after injury. We also consider several exciting new interventions that allow axons to regenerate beyond the glial scar, and discuss the implications of this work for the future of regeneration biology.
    Experimental Neurology 01/2014; 253. DOI:10.1016/j.expneurol.2013.12.024 · 4.70 Impact Factor
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    • "Using a thin needle to sever the axons, they discovered that after a period of acute degeneration of both the proximal and distal tips, the proximal axons often grew in the wrong direction. Therefore, it seems that ascending sensory axons fail to regenerate, at least in part, due to the absence of proper navigational cues. in addition, through live imaging of axons at the dorsal root entry zone, Di Maio et al. [49] demonstrated that axons may regenerate across this border into CNS territory but stall after exhibiting presynaptic features [50] . "
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    ABSTRACT: With advances in genetic and imaging techniques, investigating axon regeneration after spinal cord injury in vivo is becoming more common in the literature. However, there are many issues to consider when using animal models of axon regeneration, including species, strains and injury models. No single particular model suits all types of experiments and each hypothesis being tested requires careful selection of the appropriate animal model. in this review, we describe several commonly-used animal models of axon regeneration in the spinal cord and discuss their advantages and disadvantages.
    Neuroscience Bulletin 08/2013; 29(4):436-44. DOI:10.1007/s12264-013-1365-4 · 2.51 Impact Factor
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