Journal: Gut Microbes

Article Title: Gut dysbiosis was inevitable, but tolerance was not: temporal responses of the murine microbiota that maintain its capacity for butyrate production correlate with sustained antinociception to chronic morphine

doi: 10.1080/19490976.2024.2446423

Figure Lengend Snippet: Butyrate supplementation prolongs morphine antinociception independently of curtailment of inflammation. a) Schematic of mouse paradigm where mice were fed 100 mm sodium butyrate or sodium citrate, followed either by 9-d morphine (m.s.) (10 mg/kg s.c.) ( n = 18 ea), or daily saline (0.9% s.c.) ( n = 8 ea). Mice treated daily with m.s. were assessed for baseline nociception and antinociception on days 1, 7, and 10 at 5 mg/kg and 30 minutes later at 5 + 2 mg/kg m.s. s.c. (ED 60 and ED 90 respectively, see supplemental figure S11a). Feces were collected on days 1, 5, 9, and 11 of the paradigm to generate 16S (V4-V5 regions, see methods). b). The trajectory of tolerance was visualized as a decrease in latency to tail-flick (s) (5 + 2 mg/kg s.c. m.s.) and reported as mean; error bars are SEM. Kruskal–Wallis test followed by pairwise Wilcox test for multiple comparisons were used to determine whether citrate and butyrate groups changed over time (horizontal bars) and differed from each other (asterisks above data points) (* p < .05; ** p < .01; *** p < .001). c) Antinociception (5 + 2 mg/kg s.c. m.s.) as determined on day 10 using the radiant heat tail flick assay. Data is reported as maximum possible effect (% MPE). Kruskal–Wallis test followed by pairwise Wilcox test for multiple comparisons determined significant differences between m.s. treated citrate and butyrate supplementation groups and between m.s. treatments and saline controls (** p < .01; *** p < .001). d) Change in morphine potency was assessed by comparison of linear regressions of dose responses (ED 60 and ED 90 doses) reported as %MPE on day 1 (open squares) and day 10 (closed squares). Outer dashed lines represent 95% confidence intervals of the linear regressions and error bars represent SEM. Shift in ED 50 from day 1 to day 10 extrapolated from these regressions is reported in text. e) Day 10 serum levels of LPS binding protein (LBP) of mice supplemented with citrate (orange) or butyrate (purple) and their saline controls. LBP upper normal range (dashed line), as indicated by manufacturer, is marked for reference (Invitrogen, Waltham, MA). f) Day 10 serum levels of pro-inflammatory cytokines (IL-1β, IL-6, and tnf-α) of mice supplemented with citrate (orange) or butyrate (purple) and their saline controls. Dashed lines represent normal ranges of serum cytokine levels (50 pg/mL: IL-1β, IL-6; 150 pg/mL: TNF-α). Kruskal–Wallis test followed by pairwise Wilcox test for multiple comparisons determined significant differences between m.S. treated citrate and butyrate supplementation groups and between m.S. treatments and saline controls (* p < .05).

Article Snippet: LBP upper normal range (dashed line), as indicated by manufacturer, is marked for reference (Invitrogen, Waltham, MA). f) Day 10 serum levels of pro-inflammatory cytokines (IL-1β, IL-6, and tnf-α) of mice supplemented with citrate (orange) or butyrate (purple) and their saline controls.

Techniques: Saline, Tail Flick Test, Comparison, Binding Assay