Elsevier

Experimental Neurology

Volume 242, April 2013, Pages 11-17
Experimental Neurology

Review
In vivo imaging: A dynamic imaging approach to study spinal cord regeneration

https://doi.org/10.1016/j.expneurol.2012.07.007Get rights and content

Abstract

Upon spinal cord injury, severed axons and the surrounding tissue undergo a series of pathological changes, including retraction of proximal axon ends, degeneration of distal axon ends and formation of a dense fibrotic scar that inhibits regenerative axonal growth. Until recently it was technically challenging to study these dynamic events in the mammalian central nervous system. Here, we describe and discuss the recently established genetic tract tracing approach of in vivo imaging. This technique allows studying acute pathological events following a spinal cord lesion. In addition, the novel development of chronic spinal cord preparations such as the implanted spinal chamber now also enables long-term imaging studies. Hence, in vivo imaging allows the direct observation of acute and chronic dynamic degenerative and regenerative events of individual neurons after traumatic injury in the living animal.

Introduction

The wiring and complex connections of neuronal pathways within the mammalian central nervous system (CNS) have long been the subject of intense investigations in spinal cord research. Neuronal tracing techniques constitute indispensable tools to study the development and trajectories of axonal tracts, as well as the regeneration potential of axons after traumatic injury. Spinal cord injury (SCI) leads to a cascade of diverse pathological events: While proximal axon ends retract and do not regenerate upon injury, distal axon ends undergo a process called Wallerian degeneration (reviewed in Waller, 1850, Coleman and Freeman, 2010). At the same time meningeal fibroblasts and astroglia, as well as diverse immune cells infiltrate the lesion site and lead to restructuring of the injured tissue and formation of a dense scar (reviewed in Silver and Miller, 2004). The sequence and timing of pathological as well as regenerative events can be most reliably studied in the natural environment of the affected neurons. Hence, progress in understanding the underlying pathobiology of these complex events also depends on our ability to image individual neurons.

Classical tracing approaches are useful applications for examining anatomical relationships between various brain regions and characterizing the architecture of arborizations and synaptology of axon terminals. However, there are some limitations in using these approaches for the assessment of axonal regeneration after SCI (reviewed by Kobbert et al., 2000, Raju and Smith, 2006, Schofield, 2008): (1) Classical tracing techniques are generally “static” end-point experiments, where each animal provides only one piece of data. The adequate interpretation and analysis of the data end-points must be deduced by comparing the individual static images. (2) The tracer application requires complex surgery and extensive expertise from the scientist, potentially introducing new variables and complicating subsequent interpretation of the data. (3) The tracing of specific neuronal pathways may be incomplete or erroneous. For example, variability in amount and location of dye injection can lead to variations in the number of labeled fibers (Bareyre et al., 2005, Joosten et al., 1987, Steward et al., 2004). Some common tracers such as cholera toxin subunit B (CTB) and biotinylated dextran amines (BDA) can erroneously be taken up by fibers of passage, which have been damaged as a consequence of the dye injection (Brandt and Apkarian, 1992, Reiner et al., 2000). (4) Finally, using classical tracing approaches it is technically more demanding to label and follow single nerve fibers. Even though classical tracing techniques as well as reductive approaches, such as cell culture studies have provided useful insights into understanding axonal pathobiology after SCI, the necessity for the development of more innovative and dynamic imaging approaches in vivo was inevitable.

As one of the first examples of in vivo imaging, the genetic tract tracing technique developed by Kerschensteiner et al. (2005) allows the in situ observation and analysis of lesioned axons at the single cell level in their natural habitat. A single structure is followed over time and multiple data points are obtained from one animal. Hence, interpretation of data is easier to infer, since the pathological events may directly be observed while they are happening in the living animal. Therefore, in vivo imaging enables direct observation of dynamic degenerative and regenerative processes after traumatic injury and constitutes a unique tool to unravel the events underlying spinal cord pathology. In the future, in vivo imaging will help to gain insight into how neurons in the nervous system change in relation to behavioral adaptations, experience and especially pathological events such as SCI.

In this review we aim to describe and discuss this dynamic imaging technique. First, we delineate how a basic in vivo imaging experiment is implemented in practice. Second, we discuss the strengths of in vivo imaging and illustrate the necessity for its development in addition to conventional tracing approaches. We then address the current limitations and caveats of in vivo imaging. Finally, we suggest potential future applications of this technique.

Section snippets

The experimental procedure

In this section, we delineate the principal steps to perform an in vivo imaging experiment. This description is important for understanding the main strengths and limitations of this technique addressed in the respective sections of this review. A basic setup for in vivo imaging is depicted in Fig. 1. However, the setup is versatile and can be easily modified and customized to meet the particular researcher's needs.

Among the most useful mouse models for in vivo imaging are transgenic mice

Strengths of in vivo imaging

In vivo imaging is a dynamic imaging approach, in which a single structure, for instance a severed axon, can be followed over time. Dynamic approaches can be subdivided into two categories: continuous real-time imaging and repetitive imaging. While the former approach is most suited to study acute pathological events directly after SCI with high temporal resolution, the latter approach allows the analysis of changes that extend over a longer time frame (> 1 day). Compared to static techniques,

Limitations of in vivo imaging

Despite its numerous advantages, in vivo imaging is a fairly novel approach with diverse procedural limitations. The introduction of further technical refinements as well as the continuous development of modern microscopy approaches will steadily improve this technique and finally overcome the major drawbacks. Until then, some limitations which are inherent to in vivo imaging as well as limitations common to both in vivo imaging and classical tracing techniques need to be considered while

Perspectives

To this point, regeneration studies of the mammalian CNS have been dominated by static end-point measurements. New model systems of spinal cord regeneration will increasingly complement these established models. In particular, dynamic imaging approaches like in vivo imaging, that are adapted to observe and assess pathological events following SCI while they are occurring in the living animal will be of great value. Most importantly, in vivo imaging may now be applied to monitor injured axons

Acknowledgments

We thank Robert Schorner from the Max Planck Institute of Neurobiology in Martinsried (Germany) and Kerstin Landwehr for their assistance with the graphical work. This work was supported by the Deutsche Zentrum für Neurodegenerative Erkrankungen and the Deutsche Forschungsgemeinschaft. C.L. is a student of the International Max Planck Research School for Molecular and Cellular Life Sciences in Munich (Germany).

References (33)

  • D.M. Basso et al.

    Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains

    J. Neurotrauma

    (2006)
  • M.P. Coleman et al.

    Wallerian degeneration, wld(s), and nmnat

    Annu. Rev. Neurosci.

    (2010)
  • W. Denk et al.

    Two-photon laser scanning fluorescence microscopy

    Science

    (1990)
  • A. Di Maio et al.

    In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border

    J. Neurosci.

    (2011)
  • C. Dray et al.

    Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord

    Proc. Natl. Acad. Sci. U. S. A.

    (2009)
  • A. Erturk et al.

    Disorganized microtubules underlie the formation of retraction bulbs and the failure of axonal regeneration

    J. Neurosci.

    (2007)
  • Cited by (0)

    View full text