Multidimensional long-term time-lapse microscopy of in vitro peripheral nerve regeneration.

Abstract

In order to test the effectiveness of a new advanced time-lapse microscopy imaging and image processing and analysis system, and to do quantitative and qualitative temporal analyses of in vitro peripheral nerve regeneration, long-term time-lapse imaging of cultures of mouse dorsal root ganglia (DRGs) was performed. DRGs were placed in a Petri dish, covered with collagen gel, their attached peripheral nerves were cut in the middle, creating a gap, and the dish was filled with culture medium. Six preparations were kept on the time-lapse imaging system, which provides a suitable incubation environment and enables to capture images from multiple coordinates at x,y,z axes at desired time intervals for 13 days. In general, the time-lapse imaging system proved quite stable and efficient, although some improvements are certainly required. Two main components of peripheral nerve regeneration, outgrowth of axons and activities of resident cells, were examined. Axons started to grow during the first hour of incubation with a 16.5 microm/h rate and showed the slowest rates (0.7 microm/h) on days 8 and 9, after which they resumed higher speeds again. The first cell came out of the proximal end of the cut nerve on the second day and it was a Schwann cell (SC), which was the prominent cell type in the preparations throughout the experiment. SCs were higher in number (83.15% of all cells) but slower in migration (3.4 vs. 7.3 microm/h, P < 0.001) than other cells. Other observed characteristics of axonal outgrowth and cellular activity and interactions between axons and the cells are discussed.