gms | German Medical Science

Objective: Peripheral nerves hold the potential for regeneration. However, successful axonal regrowth can be judged only by assessing function after complete tissue regeneration taking months. We tested label-free multiphoton microscopy for its ability to permit monitoring of regeneration that precedes successful functional recovery.

Methods: The morphochemistry of the injured axolotl sciatic nerve was imaged by combining different label-free multiphoton modalities at multiple time points after transection of the axolotl sciatic nerve (0, 2, 7, 14, 21, 28, 42, 100, 120 days, n=10 for each time point). Functional recovery of the same animals was assessed by scoring the range of movement of the hind leg during swimming.

Results: Upon lesioning, we found profound changes within the area of injury. It was characterized by a strong increase in the number of two-photon excited fluorescence (TPEF) positive cells that were identified as macrophages and activated microglia by the comparison with the immunhistochemical staining for Iba1. Quantitative analysis revealed a significant increase of the number of TPEF-positive cells to maximal inflammation 28 d after injury (uninjured control: 1.0±0.2; 28 d post injury: 98.6±54.3, P<0.001). Coherent anti-Stokes Raman scattering (CARS) addressed the vibration of CH bonds and visualized changes in axonal myelin and accumulation of lipid debris. The CARS signal intensity in the lesion was decreased until 42 d after injury (uninjured control: 2.25±0.04; 42 d post injury: 1.59±0.049 P<0.001), in agreement with the immunhistochemical staining for myelin basic protein (MBP). However, CARS detected weakly/non-myelinated axons within the former lesion site in all animals 21 d after injury, which corresponded to the onset of functional recovery. In a double injury model that displayed delayed functional recovery, those weakly myelinated axons within the lesion were not confirmed until a later stage (42 days). In contrast, the time course of inflammation assessed by TPEF was not changed in the double injury model. The hind limb function was completely restored at 100 and 128 days for the standard and double injury model, respectively, and the CARS signal showed an intact sciatic nerve morphology with aligned myelin structure.

Conclusion: We envision CARS imaging as predictive monitoring tool for nerve injuries enabling early identification of lacking regeneration or therapeutic failures, allowing therapy adjustments in humans