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Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: Sensory neuron dysfunction in orthotopic mouse models of colon cancer

Fig. 5

Mitochondrial function, Ca2+ homeostasis and spontaneous activity of lumbar DRG neurons. a–c Seahorse mitochondrial function assay on DRGs collected 3 weeks after inoculation (n = 3 biological replicates/group). All values are normalized to mitochondrial-dependent respiration and the protein load. a, b Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are measured prior to treatment with oligomycin (complex V inhibitor) to calculate proton leak and ATP production (c). Maximal respiration is measured post-addition of the uncoupler FCCP. Values post-rotenone/antimycin A (complex I/III inhibitors) reflect non-mitochondrial oxygen consumption. This ‘background’ oxygen consumption is subtracted from the values in c. *: p < 0.05; **: p < 0.01, two-tailed unpaired t test. d, e Live-cell Ca2+ imaging on DRGs collected 3 weeks after MC38 inoculation. Number of measured cells: n = 78 and n = 138 for the vehicle and MC38 groups, respectively, from 2 biological replicates/group. Lumbar and non-lumbar DRG neurons were cultured and tested separately for extra control (additional Fig. 1). d DRGs from MC38-injected mice show significantly lower resting/baseline [Ca2+]i compared to vehicle-injected mice. The columns depict the average values of the 60-s baseline measurements for each group (***: p < 0.001, two-tailed unpaired t test). e Traces (average traces in bold) showing changes in intracellular [Ca2 +]i after superfusion of 25 mM potassium chloride (KCl) for 15 s. f No significant change in F340:F380 amplitude induced by KCl in e (two-tailed unpaired t test). g Multielectrode array analysis of DRG neurons from naïve mice with or without the addition of MC38-conditioned media (n = 8–10 wells/group, from at least 3 different cell cultures). Neuronal activity was measured every 4 h, for 24 h. Conditioned media or control solution (unconditioned MC38 media) was added after the 4 h measurement (marked with a red arrow and line). The addition of MC38-conditioned media was associated with a significant decrease in the spontaneous activity of DRG neurons at 20 h in vitro (*: p < 0.05 vs control, Mann–Whitney U test)

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