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    In vivo imaging analysis of the regeneration failure of dorsal root axons in adult mice

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    Genre
    Thesis/Dissertation
    Date
    2014
    Author
    Skuba, Andrew
    Advisor
    Son, Young-Jin
    Barbe, Mary F.
    Committee member
    Son, Young-Jin
    Barbe, Mary F.
    Gallo, Gianluca
    Kim, Seonhee
    Smith, George M.
    Ramirez, Servio H.
    Department
    Cell Biology
    Subject
    Cellular Biology
    Neurosciences
    Dorsal Root Entry Zone
    In Vivo Imaging
    Microscopy
    Regeneration
    Sensory Nerve
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/3580
    
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    DOI
    http://dx.doi.org/10.34944/dspace/3562
    Abstract
    After injury, dorsal root (DR) axons regenerate in the peripheral nervous system (PNS), but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the central nervous system (CNS). Examination of the dynamic axon regeneration that occurs following injury to the DR provides the opportunity to advance our understanding of what happens to sensory axons as they approach and arrive at the DREZ and expands our knowledge of sensory axon regeneration failure at the entrance to the spinal cord. Additionally, findings from these studies may offer potential avenues to provide insight into regeneration failure elsewhere in the central nervous system. Nevertheless, our understanding of the cellular and molecular processes underlying the failure of DR axons to regenerate through the DREZ is incomplete. The goal of my thesis work was to determine whether application of the time lapse-in vivo imaging technique is feasible and useful in studying dorsal root regeneration. I have also applied recently developed post-mortem analyses to the axons monitored in vivo, which provided additional insights into the mechanisms that prevent axon regeneration at the DREZ. Results in Chapters 2 and 3 demonstrate that wide-field microscopy is indeed feasible and useful for monitoring regenerating sensory axons immediately before, during, and in the days to weeks after lumbar (L5) DR crush. I was surprised to find that most axons were immobilized abruptly and chronically at the CNS portion of the DREZ, with their axon tips and shafts exhibiting features of differentiated nerve terminals. This observation raises the possibility, which has not been appreciated previously, that DR axons stop at the DREZ because their regeneration is terminated prematurely by forming synaptic contacts with unidentified postsynaptic cells. To confirm the immobilization of DR axons at the DREZ, I applied two-photon microscopy to examine the axon behavior at the DREZ at high resolution. Results described in Chapter 4 confirm those obtained with the time-lapse imaging performed with wide-field microscopy: axons arrested soon after their arrival at the DREZ did not exhibit even subtle movements. Light microscopic analyses of the failed axon tips monitored in vivo demonstrated that almost all axons stopped at the CNS territory of the DREZ, and that axon tips and adjacent shafts intensely immunolabeled with synapse markers. Ultrastructural analyses revealed that numerous axonal profiles had the characteristic features of pre- but not postsynaptic endings. Findings from these studies lead us to speculate that most, if not all, dorsal root axons become arrested as they enter the CNS territory of the DREZ by forming presynaptic terminals on non-neuronal cellular elements that differ from the dystrophic-like endings formed by a few axons. In the chapter 5, I discuss what I have found to be the key factors for successful monitoring of regenerating dorsal root axons in living animals; the feasibility, usefulness and limitations of the available techniques and future directions for studying spinal root injury and regeneration. My thesis work represents the first to employ in vivo imaging to study DR regeneration directly in living animals. This approach was more challenging to develop than we had anticipated but provided unexpected insights into the mechanisms preventing sensory nerve regeneration. Continuous application of the powerful in vivo imaging technique in combination with conventional analyses will elucidate critically important issues that previous static analyses could not decipher.
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