A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves
Introduction
Static imaging, the mainstay for evaluating regenerating nerves after traumatic injury is limited by its inefficiency and inability to capture the dynamism of nerve regeneration and Schwann cell (SC) migration. Static imaging modalities including light or electron microscopy, immunohistochemistry, and retrograde labeling are resource intensive since they mandate the sacrifice of numerous experimental animals at multiple time points to characterize regeneration. Due to discrepant processing techniques, the application of one technique often precludes the use of the other techniques on the same specimen further increasing the number of experimental animals required to obtain quantitative data (Myckatyn et al., 2004). Further, none of these techniques, in isolation, sufficiently evaluates all of the parameters of regeneration such as rate, pathway sampling, and discrimination of motor versus sensory axons (Redett et al., 2005, Witzel et al., 2005). Characterization of regeneration following traumatic nerve injury in experimental models requires a technique capable of quantifying regenerating axons and migrating SCs over time in the same mouse to complement, if not replace, existing static imaging techniques.
Axons and SCs constitutively expressing a fluorescent reporter are key to an evolving methodology designed to track migratory and regenerative processes over time. Recent advances in molecular neurobiology provide us with transgenic mice expressing genes encoding fluorescent proteins under neuron-specific or SC-specific promoters. Thy1-CFP(23) mice, previously used to study axonal retraction in developing and maturing motor endplates express cyan fluorescent protein (CFP) in all motor, and many sensory axons (Feng et al., 2000, Walsh and Lichtman, 2003). S100-GFP mice are distinguished by SCs that are labeled with green fluorescent protein (GFP) under the control of transcriptional regulatory sequences of the human S100B gene (Zuo et al., 2004). A double-transgenic thy1-CFP(23)/S100-GFP line is referred to in an article reviewing the behavior of terminal SCs (Kang et al., 2003), but this work is not intended to evaluate regeneration through nerve grafts following traumatic injury. The relationships between SCs and axons during nerve regeneration are of particular interest to clinicians that reconstruct traumatic nerve injuries with grafts.
To advance the study of nerve regeneration and SC–axonal relationships, new models enabling longitudinal imaging of SCs and axons both within the nerve fiber and at the neuromuscular junction are needed. We previously report that mice expressing enhanced yellow fluorescent protein in their axons (hThy1-EYFP) can be serially imaged after traumatic nerve injury but do not provide methods for quantifying regeneration, or use confocal microscopy, and do not simultaneously image SC migration (Myckatyn et al., 2004). In this paper we compare the rates of SC migration and axonal regeneration into cold preserved, acellular allografts used to reconstruct a sciatic nerve transection injury in live thy1-CFP(23)/S100-GFP transgenic mice. We hypothesize that the thy1-CFP(23)/S100-GFP murine model can be used for serial, quantitative analysis of SC migration and axonal regeneration to study injured nerves repaired with acellularized nerve grafts.
Section snippets
Transgenic mice
Heterozygous thy1-CFP(23) mice (Jackson, ME) were bred to homozygous S100-GFP mice (“Kosmos” line: gift from Dr. W. Thompson, Austin, TX, now commercially available through Jackson, ME). All SCs were diffusely labeled with GFP and the axons of 50% of the progeny were also labeled with CFP. We confirmed integration of the GFP transgene in the S100-GFP line visually by GFP expression in retinal glial cells and cutaneous Langerhans cells (Zuo et al., 2004). CFP transgene incorporation was
In vivo imaging of Schwann cell migration and axonal regeneration
By direct observation, the nerve grafts appeared black, with no GFP expression by SCs (Fig. 1A) or CFP expression by axons at the time of engraftment. Significant changes in fluorescent intensity, however, were noted over the following 10 days (Figs. 1B–E). Background fluorescence was measured at 158 ± 23 intensity units with minimal variation along the graft (Fig. 1E). Five days later, 1–2 mm of SC migration was noted infiltrating the proximal and distal graft (Fig. 2A) and axons had regenerated
Discussion
Using previously described advances in live imaging and transgenic techniques, we present a novel method for the independent and serial study of SC migration and axonal regeneration in a surgically relevant nerve graft model (Feng et al., 2000, Nguyen et al., 2002, Pan et al., 2003). We indirectly monitored constitutively labeled axons and SCs by evaluating the rate of change of fluorescent intensity over various components of the nerve graft. Moreover, the rate of regeneration was not uniform
Acknowledgments
This work was funded by the C. James Carrico Faculty Research Fellowship (2005–2007) from the American College of Surgeons, and the John E. Hoopes Academic Scholar (2006–2008) from the American Association of Plastic Surgeons both awarded to Dr. Terence Myckatyn. It was also funded by RO1-NS033406-12 awarded to Dr. Mackinnon and RO1-NS042794 awarded to Dr. Alexander Parsadanian by the National Institutes of Health.
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