Journal: bioRxiv

Article Title: Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain

doi: 10.1101/2022.08.29.505682

Figure Lengend Snippet: A) Supervised partial least square discriminant analysis of DI-HRMS of lumbar DRG isolated from untreated mice (day 0) and 1 or 7 days after intraplantar carrageenan (each dot represents a metabolic signature of lumbar DRG isolated from one mouse). B/C) Intensity of metabolites measured with DI-HRMS B) involved in generation of NAD + C) and used to measure 3-hydroxybutyrate/acetoacetate ratio as an indirect measure of mitochondrial NAD + /NADH ratio (n=4). D) Extracellular acidification rate (ECAR) in cultured DRG neurons from untreated mice (non-primed, n=22) or mice that had resolved from carrageenan-induced inflammatory hyperalgesia (primed, day 7, n=20). E) Course of PGE 2 -induced mechanical hyperalgesia after intraperitoneal injection with nicotinamide riboside (NR, 500 mg/kg) at day 7 in carrageenan-primed or non-primed mice and 15 min prior to intraplantar PGE 2 . F/G) Detection of mtROS formation in DRG neurons of untreated mice (day 0) or mice that had resolved from carrageenan-induced inflammatory hyperalgesia (day 7). mtROS was visualised by an intrathecal injection with MitoTracker Red CM-H2Xros (100 uM) that accumulates in mitochondria and generates fluorescence upon oxidation by mtROS. F) Mean MitoTracker Red CM-H2Xros fluorescence intensity in small and medium/large-diameter neurons at indicated days. Data visualized with a violin plot to show distribution of all data, median (black horizontal line) and quartiles (dotted line; small-diameter n= 700-1000 cells, medium/large-sized = 250-550 cells). G) Representative pictures of F. Neurons are visualised with neurotrace (blue; scale bar = 100 µM). H) Course of PGE 2 -induced mechanical hyperalgesia after intrathecal administration of mito-tempol (25 μg) at day 7 in carrageenan-primed or non-primed mice and 15 min prior to intraplantar PGE 2 . Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed by Student’s T-test (D), one-way ANOVA (B, C, F) followed by Dunnett’s multiple comparison test or two-way repeated measures ANOVA followed by a post-hoc Sidak’s multiple comparison test (E/H, stars indicate significance comparing primed conditions).

Article Snippet: In the CFA experiment, we measured hyperalgesia 4 hours after injection of NR. Intrathecal injections (5 μl) with NR (50 μg), myxothiazol (50 uM, Sigma-Aldrich), mito-tempol[ ] (25 μg, Sanbio) and oligodeoxynucleotide (3 μg/μl day 4, 5 and 6 after carrageenan, Sigma-Aldrich), were performed under light isoflurane anesthesia as described.

Techniques: Isolation, Cell Culture, Injection, Fluorescence

Journal: bioRxiv

Article Title: Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain

doi: 10.1101/2022.08.29.505682

Figure Lengend Snippet: A) Unsupervised principle component analysis (PCA) of whole metabolomics DI-HMRS dataset of HAP1 cells expressing ATPSc-KMT (WT), cells deficient for ATPSc-KMT (KO) and KO cells reconstituted with WT (KO + WT) or with enzyme dead mutant ATPSc-KMT (KO + E117A) B) Heat map of metabolite levels that were significantly changed between WT and ATPSc-KMT -/- cells. Blue = reduced intensity, red = increased intensity. Intensity of lactic acid and GA3P in C) HAP1 cells and D) N2A cells with and without ATPSc-KMT. E) Ratio of 3-hydroxybutyrate and acetoacetate which was measured with DI-HMRS and which is an indirect measure of mitochondrial NAD + /NADH balance. F/G) NAD + /NADH ratio calculated from direct measurement of cellular NAD + and NADH in F) HAP1 WT and ATPSc-KMT deficient cells and G) N2A cells after overexpression of ATPSc-KMT or control empty vector (EV). H) Course of PGE 2 -induced mechanical hyperalgesia after an intraperitoneal injection of nicotinamide riboside (NR), 15 min prior to intraplantar PGE 2 injection, in mice overexpressing ATPSc-KMT in DRG neurons. Intraplantar HSV injections were administrated at day −3 and −1 (35.000 pfu/paw) I) Same as H, but after intrathecal injection of mito-tempol, 15 min prior to intraplantar PGE 2 injection. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed by Student’s t-test (D, F and G), one-way ANOVA (C and E) or two-way repeated measures ANOVA followed by a post-hoc Sidak’s multiple comparison test (H and I).

Article Snippet: In the CFA experiment, we measured hyperalgesia 4 hours after injection of NR. Intrathecal injections (5 μl) with NR (50 μg), myxothiazol (50 uM, Sigma-Aldrich), mito-tempol[ ] (25 μg, Sanbio) and oligodeoxynucleotide (3 μg/μl day 4, 5 and 6 after carrageenan, Sigma-Aldrich), were performed under light isoflurane anesthesia as described.

Techniques: Expressing, Mutagenesis, Over Expression, Plasmid Preparation, Injection