Matrix metalloproteinase production in regenerating axolotl spinal cord

In urodele amphibian spinal cord regeneration, the ependymal cells lining t he central canal remodel the lesion site to favor axonal regrowth. We profi led the production of matrix metalloproteinases by injury-reactive mesenchy mal ependymal cells in vivo and in vitro and found that matrix metalloprote inases are involved in this remodeling process in the axolotl (Ambystoma me xicanum). The production of cell-associated matrix metalloproteinases in vi vo was shown to be identical to that in our cultured ependymal cell model s ystem. Activated and zymogen forms of matrix metalloproteinases were identi fied using zymography, chemical inhibitors of matrix metalloproteinases, an d cleavage of propeptides by organomercurials. The principal cellular prote inases consisted of matrix metalloprofeinase-2 (gelatinase A) and matrix me talloproteinase-1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials, In addition, ependymal cell conditioned medium contained secreted forms of th e enzyme undetectable in situ. Matrix metalloproteinase-9 (gelatinase B) as well as matrix metalloproteinase-2 and matrix metalloprotetnase-1 were sec reted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase-3 (prostromelysin ) secreted into the culture medium. Matrix metalloproteinases were still pr esent at 4 weeks post-lesioning when the ependymal cells have just re-epith elialized, but decreased near the completion of regeneration (8 weeks post- lesioning). Zymography showed no detectable matrix metalloproteinases in un lesioned cord but the presence of tissue inhibitor of metalloproteinase-1 i n intact cord was seen by Western blotting, This study shows that matrix me talloproteinases are associated with urodele spinal cord regeneration and v alidates the use of our ependymal cell tissue culture model system to evalu ate ependymal cell behavior during spinal cord regeneration.