Abstract
Human peripheral nerves hold the potential to regenerate after injuries; however, whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. The axolotl salamander is a regenerative model where nerves always regenerate quickly and fully after all types of injury. Here, de- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. We used coherent anti-Stokes Raman scattering to visualize myelin fragmentation and axonal regeneration. The presence of axons at the lesion site corresponded to onset of functional recovery in both lesion models. In addition, we detected axonal regrowth later in the double injury model in agreement with a higher severity of injury. Moreover, endogenous two-photon excited fluorescence visualized macrophages and revealed a similar timecourse of inflammation in both injury models, which did not correlate with functional recovery. Finally, using the same techniques, axonal structure and status of myelin were visualized in vivo after sciatic nerve injury. Label-free imaging is a new experimental approach that provides mechanistic insights in animal models, with the potential to be used in the future for investigation of regeneration after nerve injuries in humans.
Highlights
Human peripheral nerves hold the potential to regenerate after injuries; whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function
The analysis of the endogenous two-photon excited fluorescence (TPEF) predominantly shows the extent of the inflammatory response while coherent anti-Stokes Raman Scattering (CARS) imaging allowed the assessment of the overall tissue architecture, of axonal myelin and formation of lipid droplets
We confirmed that i) label-free multiphoton microscopy reveals the sequence of tissue de- and regeneration, ii) axonal regrowth shown by CARS is related to functional recovery while inflammation shown by TPEF is not; and iii) the technology is able to provide this information in situ
Summary
Human peripheral nerves hold the potential to regenerate after injuries; whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. De- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. A successful nerve regeneration requires a preserved neuronal cell body, re-growing axons crossing the site of injury and reaching the distal nerve stump that acts as guidance structure and, a correct target innervation. CARS is a non-linear variant of Raman spectroscopy and is usually applied to visualize the distribution of CHx groups in the tissue It constitutes an excellent approach for the analysis of the structure of lipid-rich myelinated axonal sheaths[12] and of intra- and extracellular lipid droplets[13]. Myelin rearrangements and inflammation are hallmarks of nerve de- and regeneration
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