Why does null hypothesis testing of serum fluorescent marker levels matter for target validation?

Null hypothesis testing of serum marker levels enables objective determination of whether interventions significantly alter intestinal permeability, supporting robust target validation and reducing false positives in early discovery.

How does isolation of the pcLoop segment fit into the discovery pipeline?

Isolating the pcLoop segment allows controlled assessment of barrier function in a defined region, facilitating mechanistic studies and enabling direct linkage between molecular targets and functional outcomes in the discovery workflow.

What do quantitative serum measurements of fluorescent markers enable in R&D?

Quantitative serum measurements provide reproducible, scalable readouts of intestinal permeability, enabling comparison across compounds, genetic models, and experimental conditions for data-driven decision making.

Why are replication requirements critical for cross-functional collaboration in permeability studies?

Replication ensures that observed changes in permeability are robust and reproducible, facilitating alignment between discovery, pharmacology, and translational teams and supporting confidence in advancing candidates.

What statistical analysis capabilities are required before implementing serum marker detection workflows?

Statistical analysis must support comparison of serum marker levels across groups, control for variability, and enable detection of significant differences, ensuring reliable interpretation and portfolio decision support.

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

JoVE Encyclopedia of Experiments

Biologie

0 weergaven • 4:00 min • July 8th, 2025

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During pathological conditions, impaired intestinal epithelial tight junction barrier causes increased permeation of harmful luminal contents into systemic circulation, resulting in increased systemic inflammation.

To assess intestinal permeability by generating an intestinal proximal colon loop or pcLoop model, prep an anesthetized mouse in the supine position. Perform a midline abdominal incision. Gently exteriorize the cecum, terminal ileum, and proximal colon, placed over wet gauze to maintain organ hydration.

Locate the proximal colon’s mesocolon — membranous folds of peritoneum containing blood vessels. Identify two sites appropriately apart in the mesocolon free of blood vessels, distal from the cecum, to be ligated. Perforate the sites.

Creating the ligature in the selected sites, make incisions close to the ligated sites to isolate a pcLoop segment without disturbing the blood supply. Flush the segment with buffer to remove any fecal matter.

Surgically tie up the cut ends of the flushed segment. Inject desired fluorescent-marker solution into the colon segment lumen. Return the organs into the abdominal cavity. Close the surgical incision and allow the mouse to recover.

Following injection, reduced intestinal barrier integrity facilitates increased paracellular transport of the fluorescent marker molecules from the intestinal lumen into the blood circulation, which can be detected in the serum fraction of blood to measure the intestinal permeability.

Prepare the mouse for surgery and exteriorize the caecum. Using wet cotton swabs, exteriorize the entire ileum and place it on top of a wet cotton gauze. Identify the proximal colon into the blood supply located in the mesocolon. Mobilize the proximal colon, and create the first ligature in an area free of vessels in the mesocolon, at about 0.5 centimeters distal from the caecum.

Measure 2 centimeters from the first ligature, and create a second ligature at an area free of blood supply in the mesocolon. Using fine scissors, carefully cut next to each ligation to isolate a 2-centimeter-long pcLoop.

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Laatst bijgewerkt: 1 augustus 2026

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

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