IL-6 is necessary and sufficient to induce injury of oligodendrocytes and axons in spinal cord organotypic cultures by generating NO. (A) CSF (100 μl) from a TM or control patient was added to culture media of spinal cord organotypic cultures, and cellular injury was assessed by ethidium homodimer uptake with Hoechst counterstain (inset). IL-6 was immunodepleted by preincubating TM CSF with an IL-6 antibody and clearing the IL-6 antibody complex with protein A sepharose. Magnification, ×20. (B) Tissue lysates from spinal cord organotypics were generated at various times after the administration of IL-6 and subjected to SDS-PAGE followed by immunoblot analysis. (C) Quantification of the data shown in B by chemiluminescent signal intensity of 3 independent experiments. (D) RT-PCR analysis of RNA derived from spinal cord organotypic cultures at the indicated times after addition of IL-6 at 2,000 pg/ml. (E) Dual-color confocal microscopy was carried out with spinal cord organotypic cultures treated with IL-6 for 24 hours. Microglia were identified by incubating live cultures with DiI-Ac-LDL, which is endocytosed by phagocytosing cells. After fixation, iNOS immunohistochemistry was carried out, revealing the expression of iNOS within microglia. Scale bar: 50 μm. (F) NT and iNOS preferentially accumulated within the exterior white matter of spinal cord organotypic cultures. Scale bar: 200 μm. (G) Addition of IL-6 to a final concentration of 2,000 pg/ml to spinal cord organotypic cultures in the presence or absence of the iNOS inhibitor 1400W. *P < 0.05. (H) WT and iNOS-heterozygous and -KO spinal cord organotypic cultures were exposed to IL-6 and assessed for cellular death.