Figure 6. Reversibility of HIV⁺ _sup ± morphine-mediated neurite damage.

Images of pre-selected neurons were captured for 24 h after initial treatments, and for an additional 48 h after treatments were changed as described in Table 1. (A) Digital images of neuronal cultures after specified time and treatments (white arrowheads indicate area of neurite outgrowth since previous image); scale bar  = 40 µm. (B) Neurons that remained alive until the experiment end (72 h) were assessed for their arborization in images taken at 0, 24 and 72 h, using Sholl analysis. The findings were reported as average Sholl scores at each time, normalized to pre-treatment (0 h) scores ± SEM. Significance was analyzed by repeated measures ANOVA and Duncan's post hoc test, from n = 45–60 neurons per treatment group (sampled from 3 separate experiments; at least 15 neurons per group per experiment). Over the period of 24 h, and in both culture systems, HIV⁺ _sup ± morphine treatments induced neurite growth arrest; in neuron-glia co-cultures, HIV⁺ _sup + morphine treatment appeared to cause neurite pruning (*p<0.05 vs. 0 h, for corresponding treatment). After removing HIV⁺ _sup at 24 h, neurite growth arrest was reversible (^$ p<0.05 vs. 24 h, for corresponding treatment); however, if HIV⁺ _sup ± morphine treatments were continued for 72 h, then neurite growth arrest was persisted. If morphine treatment continued after the removal of HIV⁺ _sup, neurite outgrowth was significantly reduced/delayed compared to neurons returned to Control_sup (^# p<0.05 vs. ‘24 h (H) then 48 h (C)’). This effect of morphine was blocked by naloxone. In the presence of glia, neurite outgrowth after removal of HIV⁺ _sup was significantly enhanced, even in the continued presence of morphine (^§ p<0.05 vs. corresponding treatment and time point in neuronal cultures; compare panels). C =  Control_sup; H =  HIV⁺ _sup (p24 = 25 pg/ml); M =  morphine sulfate (500 nM); N =  naloxone (1.5 µM).