Glial cell matrix metalloproteinase-2 generates a growth permissive nerve substrate during peripheral nerve Wallerian degeneration

Dorsal root ganglion (DRG) neurons can be cultured on cryostat tissue sections of sciatic nerve and DRG neurite outgrowth measured. In this model growing neurites travel along the Schwann cell basal lamina as do regenerating neurites in vivo. This technique, cryoculture, provided a method to explore the activity of nerve extracellular matrix (ECM) in neurite outgrowth. Matrix metalloproteinases (MMPs) have been implicated in neurite extension into matrix gels but interactions with nerve ECM have not been previously examined. DRG neurons grew longer neurites on uninjured sciatic nerve sections treated with either matrix-metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), or chondroitinase ABC, a bacterial enzyme that degrades chondroitin sulfate proteoglycan (CSPG). The effect of chondroitinase ABC mimicked that of MMP-2 and neurite outgrowth o n sections treated with MMP-2 and chondroitinase was not additive. Therefore, MMP-2 may cleave CSPG, which co-localizes with laminin in the basal lamina of peripheral nerve. Other matrix degrading enzymes including MMP-3 and bacterial hyaluronidase could not increase the neurite-promoting activity of nerve ECM. Accelerated neurite growth following enzyme treatment occurred along the basal lamina as observed by using deconvolution microscopy and double label immunofluorescence of laminin in basal lamina and GAP43 in neurites. Accelerated growth was also laminin-dependent as growth-blocking laminin antibodies totally prevent accelerated growth following enzyme treatment. To directly ascertain if MMP-2 facilitates nerve regeneration mice genetically deficient in MMP-2 were used as a source of nerve substrate and neurons for cryoculture. It is known that injured nerve that has undergone Wallerian degeneration in vivo also supports accelerated laminin-dependent neurite outgrowth in cryoculture. Therefore, sciatic nerve genetically deficient in MMP-2 was degenerated in vivo and used as a substrate for cryoculture. Degenerated, MMP-2-deficient nerve did not support increased neurite outgrowth as compared to wild-type degenerated nerve. Furthermore, treatment of degenerated, MMP-2-deficient nerve with exogenous MMP-2 restored neurite outgrowth to the length observed with degenerated, wildtype nerve. Neurons genetically deficient in MMP-2 and grown on untreated nerve sections were not shorter than wild type neurons grown on untreated sections. These observations demonstrate that during Wallerian degeneration MMP-2 generates a nerve substrate that supports accelerated neurite outgrowth.