gms | German Medical Science

Objective: The axolotl salamander exhibits an unusual regenerative capacity even after spinal cord injuries (SCI). Since various experimental approaches to treat SCI involve the application of modified biodegradable matrices, we investigated whether the frequently used substrate component alginate-hydrogel will be included in the regenerative process. The effect of the implant was analysed by label-free multiphoton microscopy (MPM).

Methods: A one millimetre long section of the spinal cord was resected and the resulting gap filled with alginate-hydrogel or left void (control). Cryosections of the injured area were prepared after 0, 2, 7, 14, 28, 56 d (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. If not stated otherwise, P-values were calculated with Welch’s t-test.

Results: MPM visualized tissue components including myelin ensheathed axons (CARS) and meninges (SHG). Upon lesioning we found profound changes within the area of injury that were characterized by an increased number of TPEF-positive cells. The Iba1 staining identified these cells as activated microglia and macrophages. The alginate-treated group showed a significantly higher number of TPEF-positive cells in all time points except for 56 d (e.g. 7 d: P<0.0001, mean: 77 and 179 cells). At 7 d post-lesioning, the formation and outgrowth of axons and meninges was significantly hindered compared to the control (P<0.0001 for both, difference of mean: 567 µm and 658 µm respectively). Growth of cells, axons or meninges into the alginate-hydrogel was not observed at any time point. However, the reconnection in the alginate-hydrogel treated animals occurred outside of the transplant and the spinal cord was successfully restored in both groups. After 56 d there were no significant differences regarding myelination (P=0.85), inflammation (P=0.24) or meningeal continuity (all values 0 µm in both groups).

Conclusion: The axolotl proved to be a promising model to evaluate the impact of novel implants on spinal cord regeneration. Through MPM meningeal, axonal and cellular behavior (e.g. inflammation or myelination) become easily assessable in unstained tissue and thus potentially in vivo. Consecutively, we will investigate novel hydrogels/implants for spinal cord regeneration and further advance our combined approach for in vivo situations.