gms | German Medical Science

Objective: A variety of SCI (spinal cord injury) models exists in vivo, but due to their complexity, comparability and clinical practicability is extremely difficult. During the last years we have established a new coculture method combining murine motor cortical (MC) and spinal cord (SC) slices. We have further investigated motor cortical regeneration and sprouting to the spinal cord and present here our data on interneurons. The impact of intraspinal networks of interneurons on recovery after incomplete sensory or motor SCI has long been known (Flynn et al., 2011). One of the major factors here is that damaged axons from the MC form new connections with the help of interneurons at the severed level with connections to sublesionel level.

Methods: MC prepared from postnatal Bl/6.GFP P0-P3 pups was cut along the coronal axis. SC was dissected from postnatal C57Bl/6 P0-P3 pups and subsequently chopped along a sagittal longitudinal plane. Thereafter, the medial MC zone was oriented to the rostral end of the SC and incubated up to 28 days. Using different approaches, we monitored interneuron in our model. On the one hand, we used transgenic mice expressing enhanced GFP under the control of the parvalbumin promotor (Pvalb-EGFP) and monitored their migration. On the other hand, we verified those migrating interneurons with additional immunohistochemical stainings using GAD67 and GAD65/67. Besides we performed electrophysiological analyses and studied the [Ca²⁺]_i response.

Results: After seven days in vitro we detected, using fluorescent live imaging, Pvalb-EGFP interneurons that migrated up to 300 µm into the wild type tissue. We observed typical interneuronal soma and dendrites. We verified those migrating interneurons with additional immunohistochemical stainings and verified a high distribution of GAD67 in our slice model. While studying the [Ca²⁺]_i response, we observed a second, traveling [Ca²⁺]_i wave following the initial response after cortical stimulation.

Conclusion: We describe further our GFP cytoarchitecture-preserving slice coculture technique to analyse regeneration between MC and SC ex vivo. Propriospinal neurons contribute to plastic reorganisation of spinal circuits (Flynn et al., 2011). Since we can monitor in our model not only the regenerating corticospinal tract, but Pvalb positive interneurons as well, we can study their role too. If the observed interneurons can integrate themselves into the existing interneuronal network by forming functional connections, they can help to detour the lesion side by forming connections with neurons with branches to sublesional level, thus creating a neuronal circuit surrounding the scar and allowing the passage of information to segments beyond it (Tuszynski and Steward, 2012).