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: 6.75). 03/2011; 31(12):4569-82. DOI: 10.1523/JNEUROSCI.4638-10.2011
Source: PubMed

ABSTRACT 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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    • "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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    Experimental Neurology 01/2014; 253. DOI:10.1016/j.expneurol.2013.12.024 · 4.62 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 · 1.83 Impact Factor
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    • "Otherwise, animals were killed by cervical dislocation under deep anaesthesia once window clarity was impeded mainly due to tissue growth between the spinal cord and Kwik-Sil or the formation of bubbles between the Kwik-Sil and the window. Similar to other studies we observed no detrimental effects of frequent repeated anaesthesia (Butterfield et al. 2004) or fluorophore injection (Dray et al. 2009; Di Maio et al. 2011). A tunable femtosecond pulsed laser (Mai-Tai, Spectra Physics, ´ Evry, France) was coupled to a Zeiss two-photon microscope (LSM 7 MP) equipped with a 20× water immersion objective lens (NA = 1.0) and five non-descanned detectors. "
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    ABSTRACT: Repeated in vivo two-photon imaging of adult mammalian spinal cords, with subcellular resolution, would be crucial for understanding cellular mechanisms under normal and pathological conditions. Current methods are limited because they require surgery for each imaging session. Here we report a simple glass window methodology avoiding repeated surgical procedures and subsequent inflammation. We applied this strategy to follow axon integrity and the inflammatory response over months by multicolour imaging of adult transgenic mice. We found that glass windows have no significant effect on axon number or structure, cause a transient inflammatory response, and dramatically increase the throughput of in vivo spinal imaging. Moreover, we used this technique to track retraction/degeneration and regeneration of cut axons after a ‘pin-prick' spinal cord injury with high temporal fidelity. We showed that regenerating axons can cross an injury site within 4 days and that their terminals undergo dramatic morphological changes for weeks after injury. Overall the technique can potentially be adapted to evaluate cellular functions and therapeutic strategies in the normal and diseased spinal cord.
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