FIG. 3.

The mitochondria of DRG sensory neurons exhibited lower respiratory chain activity. A: Oxygen consumption was assessed in freshly isolated mitochondria from lumbar DRG of age-matched control and 22-week-old diabetic rats using an OROBOROS oxygraph 2k. Coupled respiration rates were measured in the presence of pyruvate (P) (10 mmol/l), malate (M) (5.0 mmol/l), and ADP (2.0 mmol/l). The addition of FCCP (0.5 μmol/l) permits a measure of uncoupled respiratory chain activity. Addition of ascorbate (Asc) (5.0 mmol/l) and TMPD (0.5 mmol/l) permit an analysis of complex IV activity that was verified by specific inhibitors. Values are mean ± SEM; n = 5. *P < 0.05 vs. controls; **P < 0.001 vs. controls. B: Images of fluorescence confocal microscopy using TMRM in live cultures of DRG neurons isolated from control adult rats showing effect of antimycin A and oligomycin. C: Trace of TMRM fluorescence signal in the axons of cultured DRG neurons isolated from age-matched controls and STZ-diabetic rats. D: Shows the area under the TMRM fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. The area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). Values are the means ± SEM, n = 65–80 axons; *P < 0.001 compared with control, t test. The TMRM trace was characterized by nonlinear regression (one phase exponential decay). The rate constant of decay (K) = 0.013 ± 0.0004 (control) and 0.006 ± 0.0001 (diabetic). Half-life of decay = 54.19 s (control) and 108.7 s (diabetic). The Fisher parametric (F) ratio = 409.5, P < 0.0001, control vs. diabetic. The F ratio compares the goodness-of-fit of the two curves. The red arrow indicates point of injection of antimycin A + oligomycin. (A high-quality digital representation of this figure is available in the online issue.)