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Depletion of murine gut-microbiota with broad-spectrum antibiotic gavage alters intestinal motility, secretion, mucosal barrier function, immune homeostasis, energy harvest capacity and sleep architecture The glymphatic system, a perivascular circulatory system active during sleep phases and the recently uncovered meningeal lymphatic vasculature have brought into question the long standing notion of immune privilege of the central nervous system. Furthermore, within the last decade there has been a growing appreciation for the importance of coevolved, host-microbial interactions in shaping brain development and behavior. These bidirectional signaling interactions between the microbiome, the gastrointestinal tract and the brain (referred to as the microbiota-gut-brain axis) are regulated at neural, hormonal, and immunological levels. Because antibiotics are among the most commonly prescribed drugs used in human medicine, the aim of the study was to investigate relationships between antibiotics-induced dysbiosis and sleep behavior, relayed through microbiota-gut-brain axis pathways. INTRODUCTION Day: 6am/6pm Record Sleep/Wake Behavior Cages Fit With Infrared Cameras EXPERIMENTAL DESIGN 15 week old male C57BL/6 mice (N=10) Sham H20 Gavage Broad-Spectrum Antibiotic Gavage Control (N=5) (0.01ml/g BW Autoclaved H20) Mice were sacrificed after 14 days of antibiotic or sham treatments and their blood and organs were promptly harvested Once Daily 14 Days Once Daily 14 Days Antibiotic Treatment (N=5) 0.01 ml/g BW antibiotic cocktail consisting of 10mg/kg vancomycin, 25 mg/kg ampicillin, 25mg/kg neomycin, 25 mg/kg metronidazole Jonathan Lendrum, Bradley Seebach, Barrett Klein, Sumei Liu Department of Biology, University of Wisconsin-La Crosse, La Crosse, WI 54601 Correspondence: [email protected] RESULTS Broad-Spectrum Antibiotic-Gavage Altered Metabolism • Increased fecal output • Increased fecal gross energy content • Sleep impairment Immune Impairment • Decreased WBC • Reduced spleen mass • Mucosal barrier dysfunction • Enlarged caecum • Imbalance of bacterial species richness and abundance ANTIBIOTIC TREATMENT HIGHLIGHTS Figure 1. Antibiotic treatment reduced the frequency of bacteria belonging to the Bacteroidetes phylum (Bacteroidaceae family) and Firmicutes phylum (Lachnospiraceae family) (A). Phylogenetic diversity (PD whole tree) reductions indicating induction of a dysbiotic state in the antibiotic group are depicted in the rarefaction curve (B), which compares observed species richness (y-axis) against the number of sequences per sample obtained (abundancy, x-axis). Broad-Spectrum Antibiotic Gavage Induces Intestinal Dysbiosis Antibiotic Gavage Mimics Histological Characteristics of Germ-Free Mice Figure 3. Comparison of gross anatomic ceca (C and D) and photomicrographs of hematoxylin and eosin stained histological sections (A and B) of ceca from control and antibiotic-treated animals. Antibiotic treated animals show macroscopically enlarged ceca (D) and histological damage of the ceca characterized by the loss of mucosal architecture, expansion of the lamina propria and enterocyte hyperplasia (B). Control Caecum Antibiotic Caecum Figure 4. (A) WBC count and spleen mass are significantly reduced, indicating an impaired immune state in antibiotic treated animals. (B) Fecal output and caecum mass are significantly increased, while GI transit is decreased. (C) Bomb calorimetry of fecal samples shows increased fecal gross energy content, likely due to the depletion of microbes capable of fermenting indigestible sugars. Antibiotic Gavage Alters Intestinal Motility, Immune Homeostasis and Fecal Gross Energy Content CONCLUSION ACKNOWLEGEMENT Supported by: NIH R15 DK097460-01A1 (SL) and UW-L undergraduate research grant (JL). The authors thank the University of Wisconsin-Biotechnology Center DNA Sequencing Facility for providing sequencing and bioinformatics services. The results of this study suggest that broad-spectrum antibiotic-induced dysbiosis reduces gastrointestinal motility, enhances colonic ion secretion, impairs mucosal barrier function, reduces energy harvest capacity from colonic content, alters sleep behavior and may contribute to congruent neurological and gastrointestinal disorders via dysfunctional microbiota-gut-brain axis pathways. 0 10 20 30 40 50 60 70 80 90 7-9am 9-11am 11-1pm 1-3pm 3-5pm 5-7pm 7-9pm 9-11pm 11-1am 1-3am 3-5am 5-7am Mean % of Sleep Control Antibiotic Perturbation of Gut Microbiota Alters Sleep Behavior Antibiotic Control Light Dark Experimental Biology 2016 B688 1027.6 Figure 2. Intestinal permeability was measured by Ussing chamber techniques. (A) Transepithelial electrical resistance (TER) was measured 30 min after mounting the proximal colon as an assessment of intestinal permeability. (B) Horseradish peroxidase (HRP) (44 kDa) was used as a probe to examine the effects of antibiotics treatment on intestinal macromolecular permeability. (C) Short circuit current was measured to assess levels of colonic secretion.* P<0.05; ** P<0.01 compared to control. Perturbation of Gut Microbiota Increases Intestinal Permeability and Colonic Secretion Abnormal Gut Function • Increased intestinal permeability • Increased colonic secretion • Reduced gastrointestinal transit Intestinal Dysbiosis Cages Fit With Infrared Cameras Night: 6pm/6am A B ** C D A B Harlan T.D. 08806i Low Fat (10% kcal) Diet C B A ** * ** * ** ** ** Figure 5. 24-hour sleep profile of antibiotic treated animals exhibits a statistically significant reduction in the mean % of sleep during the dark phase of day (6pm-6am). Paired sample t-tests were analyzed for light and dark phases separately. The dark phase p-value <0.01** with a 14.2% difference of means.
