Article
Regeneration after sciatic nerve injury in the axolotl visualised by label-free multiphoton imaging
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Authors
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Joana Wohlers
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden
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Ortrud Uckermann
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden
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Robert Later
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden
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Andrea Meinhardt
- CRTD, DFG Research Center for Regenerative Therapies, Technische Universität Dresden
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Roberta Galli
- Klinisches Sensoring und Monitoring, Medizinische Fakultät Carl Gustav Carus Dresden
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Edmund Koch
- Klinisches Sensoring und Monitoring, Medizinische Fakultät Carl Gustav Carus Dresden
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Gabriele Schackert
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden
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Elly Tanaka
- CRTD, DFG Research Center for Regenerative Therapies, Technische Universität Dresden
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Gerald Steiner
- Klinisches Sensoring und Monitoring, Medizinische Fakultät Carl Gustav Carus Dresden
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Matthias Kirsch
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden
| Published: | June 2, 2015 |
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Outline
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Objective: Advances in the treatment of nervous system injuries are limited due to the late assessment of its limited success. Nerve injury cannot be monitored until the natural course from reconnection to functional activity has succeeded. There is a clinical need for high-resolution in vivo imaging to monitor regeneration of the nervous system. Label-free multiphoton techniques have the ability to close this gap and to advance imaging based diagnostics.
The purpose is to characterize regeneration of the peripheral nervous system in a highly regenerative model, namely the axolotl salamander.
Method: In this project we created a transection of the sciatic nerve in the axolotl. The neurological status of the animals was assessed using a swim analysis to obtain the axolotl sciatic functional index (ASFI).
Animals were euthanized on day 0, 2, 7, 14, 21, 28 and 99 after injury (n=10 per group, total n=70). Cryosections were analysed with multimodal multiphoton imaging (MMP). Second harmonic generation (SHG) was used to display collagen, two photon excited fluorescence (TPEF) probed endogenous fluorophores and coherent anti-stokes Raman scattering (CARS) tuned on CH2 vibration visualized lipid-rich structures. Immunofluorenscence staining served as a reference using antibodies against myelin basic protein (MBP) for myelin sheaths, neurofilament (NF) and beta-III-tubulin (β3T) for axons and Iba1 for microglia.
Results: MMP imaging was able to classify and visualize various normal and injured tissue stages in the sciatic nerve. Upon lesioning, we found profound changes within the area of injury which was characterized by a strong increase of the TPEF signal that was assigned to macrophages and activated microglia by the comparison with the Iba1 signal. The CARS signal intensity decreased distal to the lesion, which correlated well to the diminished MBP signal and represented degeneration of the nerve tissue. These results corresponded to the impaired mobility that was revealed by in the swim analysis at the respective time points. After 99 days the function was completely restored and the CARS and MBP signal confirmed an intact sciatic nerve morphology with aligned myelin structure.
Conclusions: MMP imaging allowed to follow injury induced alterations in the sciatic nerve, in particular to follow temporal changes of the myelination status. Consecutively, we will investigate degeneration and regeneration as well as therapeutic interventions in vivo with special attention to the myelination process.
