THE INFLUENCE OF THE MICROENVIRONMENT ON NEURITE REGENERATION IN VITRO (RETROGRADE RESPONSE, SYMPATHETIC NEURON, AXON)

Long term organ cultures of embryonic rat superior cervical ganglion were employed as a model to study one, the influence of the physico-chemical culture environment on the rate and pattern of neurite outgrowth, two, the effect of axonal injury on the retrograde response and, three, the neuronal response to a second nerve fiber injury. In this study, cultures were fed with L-15 medium containing 5 mM glucose, 10 ng/ml NGF and 0.1% FBS. The reduced serum concentration in the culture medium reduced the number of non-neuronal cells in the neuritic field thereby facilitating the assessment of rate and pattern of neurite outgrowth. The reduced serum levels enhanced the rate of neurite elongation. Uptake of (\u273)H-norepinephrine, used as a measure of neuritic sprouting, increased linearly as the neuritic field enlarged. In addition, an inverse, temporal correlation between rate of neurite extension and activity of tyrosine hydroxylase (TH) was observed during the first week of explantation. TH activity declined during the first four days of culture. After this time, activity increased and reached day 0 control levels by day six. Concurrent with this reversible change in TH activity is an increase in the rate of neurite outgrowth during the first four days of explantation. After day 4, the rate declines. The pattern of and rate of neurite outgrowth was found to vary according to the type of substratum. On collagen, neurites grew in thin fascicles. Neurite fasciculation increased on fibronectin, uncoated tissue culture plastic and Primaria. In contrast to the pattern of neurite outgrowth on the other substrata tested, neurites on poly-D-lysine grew outward in a clockwise curvature around the ganglion. There was a decrease in the rate of regeneration with increasing fasciculation. To determine the effect of reduced substratum area on neurite outgrowth SCG were grown in suspension cultures. Light and electron microscopy revealed an extensive neuritic growth by SCG neurons in suspension explant cultures. The effect of modifying the chemical environment of the culture on neurite outgrowth was determined. Nerve growth factor increased the uptake of (\u273)H-norepinephrine and the survival of neurons in SCG cultures. Mixed bovine brain gangliosides had no effect on neuritic regeneration at concentration of 7.5 and 75 ug/ml, as measured by (\u273)H-norepinephrine uptake. A ganglioside concentration of 750 ug/ml caused neuritic degeneration. A second retrograde response was initiated in these cultures by lesioning the neurites with the neurotoxin 6-hydroxydopamine (6-OHDA). This response was characterized by a reversible change in activity of tyrosine hydroxylase and an accelerated rate of neurite outgrowth. In addition, neurite transection also increase rate of neurite outgrowth