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Keywords:

  • chondroitin sulphate;
  • dorsal root ganglion;
  • neurite outgrowth;
  • PC12 cells;
  • proteoglycans

Abstract

The addition of chondroitin sulphate proteoglycans (CSPGs), purified from the rat brain, to the culture medium of PC12D cells inhibited their proliferation and neurite outgrowth. Therefore, we investigated the effects of several extracellular components on the inhibitory actions of CSPGs on PC12D cells, as well as their immunocytochemical distribution in the rat embryo to determine whether the findings in vitro could be reproduced in vivo. Coating of the substratum with polylysine was necessary for the appearance of the inhibitory effects of brain CSPGs on PC12D cells. The additional pretreatment of polylysine-coated dishes with laminin or fibronectin promoted the outgrowth of neurites from PC12D cells. Laminin and fibronectin, but not collagen (types I and IV) and CELL-TAK (cell adhesion molecules), prevented the inhibitory effects of brain CSPGs in a concentration-dependent manner. Doses producing 50% reduction by laminin (or fibronectin) of the CSPG effects were 1.5 (or 25) μg/ml for neurite outgrowth and 2.2 (or 28) μg/ml for proliferation. The ratio of dish-attached CSPGs to laminin necessary for 50% reduction was about ∼50:l (wt/wt). Laminin from any source had the same effect. Brain CSPGs also obviously impeded the growth of fibres from dorsal root ganglion explants and primary cultured dorsal root ganglion neurons. Neurocan (a major CSPG in the brain)-like immunoreactivity was detected in the boundary caps and roof plate in the rat embryo at 13.5 days of gestation, when DRG neurons were extending their axons to the neural tube. The distributions of laminin and tenascin appeared, respectively, to be slightly and considerably different from that of neurocan. These results suggest that brain CSPGs can generate barriers to the growth of axons from the dorsal root ganglion and that the ratio of CSPGs to laminin may be important in regulating such growth.