gms | German Medical Science

Objective: Spinal cord injury (SCI) research is lacking an in vivo imaging tool to study the therapeutic benefit and tissue reorganization in vivo. Amphibians such as the axolotl are known for their inherent unique capability of complete structural and functional recovery of central nervous tissue. For this reason it constitutes an ideal model to study the different steps of degeneration and regeneration after spinal cord injury. We used label-free multimodal non-linear optical imaging that can be applied in vivo to display time-dependent morphochemical tissue properties.

Method: We performed a spinal cord lesion in the axolotl tail located 15 myotomes caudal of the cloacae by dissecting the spinal cord without further tissue removal to ensure a reproducible lesion size. The animals were sacrificed either immediately after injury, or after day 2, 7, 14 and 28 (n= 7 per group). Cryosections were prepared and analyzed with multimodal non-linear optical imaging addressing collagen with second harmonic generation (SHG), endogenous fluorophores with two photon excited fluorescence (TPEF) and lipid structures with coherent anti-stokes Raman scattering (CARS) microscopy. For reference histology, conventional immunofluorescence staining using various antibodies such as MBP (myelin), βIIITubulin (neurons), NF (axons), GFAP (radial glia) and Iba1 (microglia) was peformed.

Results: Multimodal optical imaging was able to classify and visualize various normal tissue components including myelin ensheathed axons (CARS) and connective tissue (SHG) in the uninjured spinal cord. Upon lesioning, we found profound changes within the area of injury which was characterized by a strong increase of the TPEF signal indicating a high metabolic rate. However, the CARS signal decreased in the vicinity of the regenerating growth cones of the advancing ependymal tube which displayed a circumscribed SHG signal indicating an epithelial cover. Immunostaining correlated well with e.g. neurofilament expression at the tip of each growth cone. The varying MBP signal correlated well to the CARS intensity on both sides of the lesion.

Conclusions: Multimodal optical imaging proved to be a promising technique to follow injury induced alterations in the spinal cord, in particular to follow the temporal changes of the myelination status of the regenerating spinal cord. Consecutively, we will investigate degeneration and regeneration, as well as therapeutic interventions in vivo with special attention to the myelination process.