Artikel
Regeneration in the Axolotl nervous system visualized by label-free multiphoton microscopy
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Autoren
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Jonas Bendig
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Joana Wohlers
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Ortrud Uckermann
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Robert Later
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Gabriele Schackert
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Elly Tanaka
- DFG Forschungszentrum für regenerative Therapien Dresden (CRTD), Technische Universität Dresden, Dresden, Germany
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Gerald Steiner
- Klinisches Sensoring und Monitoring, Klinik und Poliklinik für Anästhesiologie und Intensivtherapie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
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Matthias Kirsch
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany; DFG Forschungszentrum für regenerative Therapien Dresden (CRTD), Technische Universität Dresden, Dresden, Germany
| Veröffentlicht: | 8. Juni 2016 |
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Gliederung
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Objective: Regeneration of the CNS and PNS cannot be monitored until we have a final functional readout: movement or paresis. Therefore, there is an urgent need for in vivo imaging modalities to analyse and control therapeutic impact after damage to the nervous system. We investigated time-dependent morphochemical tissue changes after CNS and PNS injury in the axolotl by label-free multiphoton microscopy (MPM).
Method: Two models of injury were investigated: spinal cord transection or sciatic nerve transection, cryosections of the injured area were prepared at 0d, 2d, 7d, 14d, 24d, 28d, 42d and 100d post-lesioning (n=10 per time point) and analyzed with MPM. Specifically, coherent anti-stokes Raman scattering (CARS) probed lipid-rich myelin via visualization of CH2 vibrations, while two photon excited fluorescence (TPEF) and second harmonic generation (SHG) displayed endogenous fluorophores and collagen, respectively. Immunostaining for myelin basic protein, neurofilament and ionized calcium binding adaptor molecule 1 (Iba1) served as histological reference.
Results: MPM was able to visualize and classify various normal and injured tissue components including myelin ensheathed axons (CARS) and connective tissue (SHG). Upon lesioning we found profound changes within the area of injury characterized by a strong increase of the TPEF signal in both models. The Iba1 staining characterized these TPEF+-cells as activated microglia and macrophages. In contrast, the pattern of myelin-related changes was different in the CNS and PNS. 21 d after sciatic nerve injury, the CARS signal intensity decreased to 92 % proximal (not significant) and to 76 % (P=0.016) distal to the lesion, indicating myelin fragmentation and degeneration in the distal part. Furthermore an invasion of foam cells was observed. After 100 days, the CARS signal indicated intact sciatic nerve morphology. In the injured spinal cord, myelin retraction was likewise observed in the cranial and caudal part until day 14. By day 28 the original structure of the spinal cord was completely restored.
Conclusions: MPM proved to be a promising technique to follow injury induced alterations in the nervous system, in particular to follow the temporal changes of the myelination status. All reconstituted lesions resulted in functional recovery implying a potential diagnostic and prognostic impact of the technique. Consecutively, we will investigate degeneration and regeneration during therapeutic interventions.
