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An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance
Published on: December 3, 2020
Small Bowel Resection Increases Paracellular Gut Barrier Permeability via Alterations of Tight Junction Complexes
Cathleen M Courtney1, Emily J Onufer1, Keely G McDonald2
1Division of Pediatric Surgery, Department of Surgery, St. Louis Children's Hospital, Washington University School of Medicine, St. Louis, Missouri.
Background:
Short bowel syndrome resulting from small bowel resection (SBR) is associated with significant morbidity and mortality. Many adverse sequelae including steatohepatitis and bacterial overgrowth are thought to be related to increased bacterial translocation, suggesting alterations in gut permeability. We hypothesized that after intestinal resection, the intestinal barrier is altered via toll-like receptor 4 (TLR4) signaling at the intestinal level.
Methods:
B6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice underwent 50% SBR or sham operation. Transcellular permeability was evaluated by measuring goblet cell associated antigen passages via two-photon microscopy. Fluorimetry and electron microscopy evaluation of tight junctions (TJ) were used to assess paracellular permeability. In parallel experiments, single-cell RNA sequencing measured expression of intestinal integral TJ proteins. Western blot and immunohistochemistry confirmed the results of the single-cell RNA sequencing.
Results:
There were similar number of goblet cell associated antigen passages after both SBR and sham operation (4.5 versus 5.0, P > 0.05). Fluorescein isothiocyanate-dextran uptake into the serum after massive SBR was significantly increased compared with sham mice (2.13 ± 0.39 ng/μL versus 1.62 ± 0.23 ng/μL, P < 0.001). SBR mice demonstrated obscured TJ complexes on electron microscopy. Single-cell RNA sequencing revealed a decrease in TJ protein occludin (21%) after SBR (P < 0.05), confirmed with immunostaining and western blot analysis. The KO of iTLR4 mitigated the alterations in permeability after SBR.
Conclusions:
Permeability after SBR is increased via changes at the paracellular level. However, these alterations were prevented in iTLR4 mice. These findings suggest potential protein targets for restoring the intestinal barrier and obviating the adverse sequelae of short bowel syndrome.
Insights
Short bowel syndrome (SBS) increases gut permeability through paracellular changes, but toll-like receptor 4 (TLR4) knockout prevents this. Targeting TLR4 may restore the intestinal barrier after small bowel resection (SBR).
Area of Science:
- Gastroenterology and Hepatology
- Immunology
- Molecular Biology
Background:
- Short bowel syndrome (SBS) following small bowel resection (SBR) leads to significant morbidity and mortality.
- Adverse effects like steatohepatitis and bacterial overgrowth are linked to increased bacterial translocation, suggesting compromised gut permeability.
- Toll-like receptor 4 (TLR4) signaling is hypothesized to mediate intestinal barrier alterations post-intestinal resection.
Purpose of the Study:
- To investigate the role of toll-like receptor 4 (TLR4) in altering intestinal barrier function after small bowel resection (SBR).
- To determine if blocking TLR4 signaling can prevent increased gut permeability following SBR.
Main Methods:
- Small bowel resection (SBR) or sham operation was performed on B6 and intestinal-specific TLR4 knockout (iTLR4 KO) mice.
- Transcellular permeability was assessed via goblet cell antigen passage using two-photon microscopy.
- Paracellular permeability was evaluated using electron microscopy of tight junctions (TJ) and fluorescein isothiocyanate-dextran uptake; single-cell RNA sequencing, Western blot, and immunohistochemistry analyzed TJ protein expression.
Main Results:
- While transcellular permeability remained unchanged, paracellular permeability significantly increased in SBR mice, evidenced by increased dextran uptake and obscured TJs.
- Single-cell RNA sequencing revealed a significant decrease in the TJ protein occludin (21%) post-SBR, confirmed by other analyses.
- Intestinal-specific knockout of TLR4 (iTLR4 KO) successfully mitigated the SBR-induced alterations in intestinal permeability.
Conclusions:
- Intestinal resection increases gut permeability primarily through paracellular mechanisms.
- These permeability alterations are prevented in mice lacking intestinal TLR4, implicating TLR4 signaling in the process.
- Targeting TLR4 signaling presents a potential therapeutic strategy for restoring intestinal barrier function and mitigating SBS complications.
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