ORGANOTYPIC SPINAL-CORD CULTURE IN SERUM-FREE FIBRIN GEL - A NEW APPROACH TO STUDY 3-DIMENSIONAL NEURITE OUTGROWTH AND OF NEUROTOXICITY TESTING - EFFECTS OF MODULATING THE ACTIN AND TUBULIN DYNAMICS AND PROTEIN-KINASE ACTIVITIES

Spinal cord explants from embryonic day seven (E7) chicken embryos wer e cultured without serum and in the presence of aprotinine, in a three -dimensional fibrin matrix. These conditions promote a robust, radial, unfasciculated outgrowth of neurites that are tipped by elaborate gro wth cones. Routinely after 5 days, the neurite outgrowth intensity (NO I) was determined by measuring the optical density of the immunostaine d neurites (image analysis program OPTIMAS version 5.2) within defined areas, extending radially for up to 3 mm from the explant border. A d ose-dependent inhibition of NOI was determined for the cytoskeleton-af fecting drugs nocodazole (half maximal inhibition ([I-50]), 0.02 mu M) , taxol ([I-50], 0.016 mu M), cytochalasin D ([I-50], 0.006 mu M), and tetramethyl lead ([I-50], 0.05 mu M). Likewise, NOI was decreased in a dose-dependent fashion by ML-9 and RO-31-8220, inhibitors of myosin- light chain kinase and protein kinase C (PKC), respectively. The addit ion of 1,2-dioctanoyl-s,n-glycerol, a potent activator of PKC, led, at 5 mu M, to an increase and at 30 and 60 mu M to a decrease in NOI. Th e described system provides a rapid, reproducible, and quantitative as say for the effects of exogenous factors on the mode and intensity of neurite outgrowth. (C) 1997 Elsevier Science Ireland Ltd.