title: Organotypic spinal cord culture in serum-free fibrin gel: a new approach to study three-dimensional neurite outgrowth and of neurotoxicity testing: Effects of modulating the actin and tubulin dynamics and protein kinase activities.
abstract: Spinal cord explants from embryonic day seven (E7) chicken embryos were 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 growth cones. Routinely after 5 days, the neurite outgrowth intensity (NOI) was determined by measuring the optical density of the immunostained neurites (image analysis program OPTIMAS version 5.2) within defined areas, extending radially for up to 3 mm from the explant border. A dose-dependent inhibition of NOI was determined for the cytoskeleton-affecting drugs nocodazole (half maximal inhibition ([I50]), 0.02 microM), taxol ([I50], 0.016 microM), cytochalasin D ([I50], 0.006 microM), and tetramethyl lead ([I50], 0.05 microM). 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 addition of 1,2-dioctanoyl-s,n-glycerol, a potent activator of PKC, led, at 5 microM, to an increase and at 30 and 60 microM to a decrease in NOI. The described system provides a rapid, reproducible, and quantitative assay for the effects of exogenous factors on the mode and intensity of neurite outgrowth.
authors.affiliation: Institute of Zoology, University of Hohenheim-Stuttgart, Stuttgart, Germany. roesnerh@uni-hohenheim.de
authors.forename: H
authors.initials: H
authors.lastname: Rösner
authors.forename: G
authors.initials: G
authors.lastname: Vacun
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D002642:Chick Embryo; D015638:Cytochalasin D / Q000494:pharmacology; D003720:Densitometry / Q000379:methods; D004075:Diglycerides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005337:Fibrin; D005782:Gels; D007211:Indoles / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009498:Neurotoxins / Q000494:pharmacology*; D015739:Nocodazole / Q000494:pharmacology; D009924:Organ Culture Techniques; D017239:Paclitaxel / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology*; D013756:Tetraethyl Lead / Q000031:analogs & derivatives / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism*
chemical_list: D000199:Actins; D001381:Azepines; D004075:Diglycerides; D004791:Enzyme Inhibitors; D005782:Gels; D007211:Indoles; D009498:Neurotoxins; D014404:Tubulin; C056218:ML 9; C044295:1,2-dioctanoylglycerol; D013756:Tetraethyl Lead; D015638:Cytochalasin D; D005337:Fibrin; D007611:Aprotinin; C014849:tetramethyllead; D011494:Protein Kinases; D017239:Paclitaxel; D015739:Nocodazole; C064758:Ro 31-8220