EFFECTS OF NERVE SEGMENT SUPERNATANTS ON CULTURED SCHWANN-CELL PROLIFERATION AND LAMININ PRODUCTION

Mouse sciatic nerves were transected and 3 hr to 16 days later proxima l segments were removed and homogenized. Supernatants of these segment s or of normal sciatic nerves were added to Schwann cells maintained i n Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FC S). After 6 days, Schwann cells were solubilized and the protein conte nt was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzyme-linked immuno sorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or fr om control nerves. Increased surface and cytoplasmic anti-laminin immu noreactivity also was found in Schwann cells treated with 24 hr supern atants. To identify the source(s) of this effect, proximal segments re moved 24 hr after transection were bisected; supernatants were prepare d from each half and tested. Significant increases in laminin producti on were produced by supernatants from both halves. When supernatants f rom proximal and distal halves were compared, the latter produced sign ificantly higher laminin levels. Electron microscopic examination of b oth halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal hal ves appeared normal. Schwann cell proliferation also was compared in s upernatant-treated cultures by using a bromodeoxyuridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regene rative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell prolife ration and laminin production. (C) 1994 Wiley-Liss, Inc.