Dissociated Neurons Regenerate into Sciatic But Not Optic Explants in Culture Irrespective of Neurotrophic Factors’ Nerve

Explants of adult or lo-day-old rat sciatic and optic nerves were implanted as “bridges” through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in viva regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF. Neurite outgrowth and regeneration in vivo and in vitro depend on a variety of epigenetic mechanisms. Pure neuronal cultures, particularly in defined media without serum or other rich additives, need specific soluble factors and substrates for optimal survival and neurite production. The best characterized soluble factor is nerve growth factor (NGF), which is necessary for survival and differentiation of sympathetic and subpopulations of sensory neurons (Thoenen and Barde, 1980). Moreover, NGF stimulates the rate of neurite outgrowth and branching frequency and influences the directionality of the growth cone movements (Gundersen and Barret, 1980; Campenot, 1982; Seeley and Greene, 1983). Other factors with different neuronal specificities have been or are being purified from various peripheral organs and from the central nervous system (CNS) (Barde et al., 1982, 1983; Barbin et al., 1984; Berg, 1984). In addition to soluble factors, the culture substrate is essential for neuronal adhesion and influences the number, shape, and growth rate of neurites (Letourneau, 1975; Manthorpe et al., 1983a; Rogers et al., 1983). Received October 5, 1984; Revised February 27, 1985; Accepted February 28, 1985 ’ We thank Mrs. Ch. Mtiller for excellent technical and photographic assistance, Drs. A. Acheson and D. Edgar for their critical review of the manuscript, and Mrs. E. Eichler and Mrs. H. Macher for their secretarial help. * To whom correspondence should be sent, at: Institute for Brain Research, University of Zurich, August Forel-Str. 7. CH 8029, Zurich, Switzerland In vivo, the crucial role of the environment through which neurites grow became evident from developmental and regeneration studies. During development, guidance of axons seems to occur in the CNS as well as in the periphery along favored substrate pathways (Katz et al., 1980; Sanes, 1983). In the developing CNS, radial glial cells and glial “bridges” play important roles (Rakic, 1982; Silver et al., 1982). During regeneration in the peripheral nervous system, Schwann cells and basement membranes are very favorable substrates for growing axons (Nathaniel and Pease, 1963; Sanes, 1983). The most impressive effect of the environment on neurite growth and elongation becomes apparent in comparing the regeneration in the CNS and the peripheral nervous system of higher vertebrates. No regeneration of lesioned axonal connections takes place within the differentiated CNS. However, a large variety of central neurons are able to produce axons which elongate over long distances inside a peripheral nerve environment (Ram6n y Cajal, 1928; Richardson et al., 1980, 1984; Weinberg and Raine, 1980). Thus, when sciatic nerves are implanted into the spinal cord or the brain, they are invaded by central axons, which, however, stop abruptly at the other end of a “bridge-transplant” when they encounter CNS tissue again (David and Aguayo, 1981). I f optic nerves are implanted into sciatic nerves they are almost completely avoided by the regenerating peripheral axons (Aguayo et al., 1978; Weinberg and Spencer, 1979), and the regeneration of dorsal root fibers after a crush of the dorsal root stops at the Schwann cell-CNS interface (Stensaas et al., 1979; Bignami et al., 1984). The molecular basis of these differences in the microenvironment between the CNS and the peripheral nervous system is largely unknown. Production of specific trophic factors by Schwann cells which induce the regenerating response of the neurons or locally support neurite growth and elongation and the absence of such factors in the adult CNS could be one determinant (Ram6n y Cajal, 1928; Kiernan, 1979; Longo et al., 1983). The presence of specific substrates favorable for axonal growth in the periphery and their absence in the CNS is another possibility. In addition, mechanical barriers, e.g., by astrocytic scar tissue, were often discussed as a reason for the lack of CNS regeneration (Ram6n y Cajal, 1928; Kiernan, 1979; Reier et al., 1983). All of the in vivo experiments performed so far do not allow one to distinguish between these different hypothetical mechanisms. In particular, it would be of importance to be able to distinguish between the “lack of trophic factor” hypothesis and the presence of a non-permissive substrate for neurite growth in the CNS. Only in vitro experiments permit the degree of control adequate to define the relative importance of these various influences. In the present study, we used dissociated neurons from neonatal rat sympathetic or sensory ganglia which are grown under optimal culture conditions in the presence of NGF and are capable of regenerating an extensive axonal network. In a chamber culture system these neurons were given the choice to grow into explants of sciatic nerves or optic nerves as “bridges” or “tunnels” to other compartments of the culture dish. By light and electron microscopy,