1. 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: Lendrum.jona@uwlax.edu
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.
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Mean%ofSleep
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
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A B
Harlan T.D. 08806i Low Fat (10% kcal) Diet
C
B
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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.