Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C14
In vivo imaging of long-term reactions of glia and neurones after lesions of the spinal cord in triple-transgenic mice with fluorescent proteins
F. Nadrigny1,2, P. Dibaj1, H. Steffens1,3, F. Kirchhoff1,4, E. D. Schomburg3
1. Neurogenetics, Max-Planck-institute for Experimental Medicine, G
Neuronal regeneration after spinal cord injury may be impeded by a glial scar, but its character and temporal development is still vague. Triple-transgenic mice with labeled projection neurons and glial cells with different fluorescent proteins (axons/YFP, microglia [MG]/GFP, astrocytes/CFP) were imaged with two photon microscopy before and after laser-induced spinal cord lesions (20-40 µm diameter at L4). The lesions were regularly re-inspected for up to one year. The initial laminectomy at spinal cord L4 and re-opening for re-inspections were performed under full volatile anaesthesia (1:1 N2O:O2 and isoflurane initially 5%, then 2%). The days after operation, the mice received buprenorphin (0.1 mg/kg per day i.p.). Within minutes after the lesion, MG sent their processes towards the lesion. During the next days, nearby MG cells migrated toward the lesion accumulating and staying there for about a week, then slowly diminishing during the next five months. Monocytes (also GFP-labeled) crossing the blood-brain barrier during the acute reaction and invading the lesion site were not found. Astrocytes were slowly activated and started to extend processes to the lesion after two days. An astroglial reaction surrounding the lesion site was fully developed after a week and subsequently decreased within five months. Within hours after the lesion, dissected axons formed bulbous debris which was partly engulfed by MG processes directed to the lesion. Other axonal bulbs remained at their place caudal to the lesion for weeks. Occasionally, axonal sprouting was detected about three months after injury. Initially, neuronal sprouts were not always straightly directed towards the lesion but later they could cross the site of injury, when the glial accumulation had almost vanished. Mechanically induced injuries evoked similar spatiotemporal cellular reactions but did not allow such a systematic investigation since they were less well defined. The combination of multi-cellular labeling with long-term imaging allowed an exploration of the time course of complex cellular responses after spinal cord injury. Detailed knowledge of the temporal behavior of the main cell types involved in the reaction to spinal cord injury may provide valuable hints for a therapeutical approach to improve axonal survival and regeneration.
Where applicable, experiments conform with Society ethical requirements