Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80% pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000, 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypeptides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000-dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining.Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000-dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not.These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics. KEY WORDS neurofilaments glial filamentsIsolation of intact intermediate filaments (8-10 nm diameter) from the mammalian central nervous system has depended on the axonal flotation technique (19), which uses the presence of the myelin around the axons to float neuronal material away from the other brain tissue. One can then remove the myelin from the axons by exposure to hypotonic solution and obtain purified intermediate filaments by applying the nonmyelin material to a sucrose gradient (22). The filamentrich fraction obtained from the gradient appears to be over 90%. 8-10-rim filaments by electron microscopic examination. They are present mostly as loose bundles resembling neurofilaments, although some tight bundles resembling glial filaments are also observed. When this fraction is J. CELL BIOLOGY 9 The Rockefeller University Press

Previous experimental studies have suggested that tumor necrosis factor (TNF) may have either a beneficial or a detrimental role in wound healing. Control and doxorubicin-treated (6 mg/kg, intravenously) rats underwent paired dorsal 5-cm linear wounds and had either vehicle or recombinant (r)TNF (0.5, 5, or 50 micrograms) applied locally to the wound. Paired wounds were harvested at 7 and 14 days after wounding and analyzed for wound-bursting strength (WBS) and activity of the gene for type 1 collagen and TNF. Doxorubicin treatment decreased WBS at 14 days but not at 7 days after wounding. Local application of 50 micrograms of rTNF decreased WBS in saline-treated rats and concentrations of 5 and 50 micrograms decreased WBS in doxorubicin-treated rats when measured 7 days after wounding. These effects dissipated when WBS was measured 14 days after wounding. Doxorubicin decreased wound collagen gene expression and local TNF treatment decreased wound collagen gene expression in saline-treated rats and further decreased it in doxorubicin-treated rats. The decrement in collagen gene expression induced by rTNF increased as the local dose of rTNF increased. The gene for TNF was not detectable in wounds from normal or doxorubicin-treated rats at 3, 7, 10, or 14 days after wounding. These data suggest that the gene for TNF is not expressed in wounds and that the local application of TNF is detrimental to wound healing as it decreases WBS and activity of the gene for collagen.

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Radiation of skin results in slower healing of open wounds, and provides an in vivo system for evaluation of topical dressings and growth factors in radiation-impaired wounds.