Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Summary

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Abstract

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

Introduction

The human gastrointestinal (GI) tract is a multiple-foot-long organ system, roughly approximated as a tube of varying dimensions and physical properties¹. As the contents move through its length, the GI tract's primary function is to absorb substances critical for life. The small intestine is specifically responsible for nutrient absorption. The small intestine's transit is tightly regulated to match the digestion and absorption functions, resulting in various motility patterns. Bayliss and Starling described the "law of the intestine"² in 1899, showing the contractile propulsion program in the intestine ....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic.

1. Setup

1. Fast 8- to 10-week-old mice for 4 h. Provide mice with access to water.
   NOTE: We use wild-type male C57BL/6J mice for all experiments presented here, but they can be performed on mice of any strain, gender and genotype.
2. Cool 15 mL of distilled water in a 50 mL conical tube in a 4 °C refrigerator.
3. Heat another 15 mL of distilled water in a beaker using a hot plate with a magnetic stir bar to approximately 80-90 °C.
4. Measure 0.5% methylcellu....

Results

We show representative outcomes from Step 3 onwards. Figure 1 shows the intact explanted bowels, with fluorescent measurements overlaid. The stomach (purple) is laid along the same axis as the small intestine (orange), but we prefer moving the cecum (blue) to the side to prevent overlap with the large intestine (orange). As evidenced in the left panel, this is not always possible due to organ size. We cut the small bowel at ~200 mm to maximize the coverage of continuous segments, but this is.......

Discussion

The GI tract, like other tubular organs, such as blood vessels, requires mechanical sensors and effectors to maintain homeostasis^26,27,28. However, the GI tract is unique in that the physical properties of the materials that traverse it are not constant across meals. Intraluminal contents of various physical properties (solid, liquid, and gas) transit the gut, generating different mechanical inputs to the GI mechanoreceptors. In.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J mice	Jackson Laboratory	664	other mice can be used with this protocol
Dissection tools	n/a	n/a
Excel software	Microsoft	n/a	used for spreadsheet analysis
Fluorescent Green Polyethylene Microspheres 1.00g/cc 75-90um - 10g	Cospheric	UVPMS-BG-1.00 75-90um - 10g	"smaller beads" in the manuscript
Fluorescent Green Polyethylene Microspheres 1.00g/cc 180-212um - 10g	Cospheric	UVPMS-BG-1.00 180-212um - 10g	"larger beads" in the manuscript
Gavage needles	Instech	FTP-18-50-50
ImageJ software	n/a	n/a	used to extract fluorescence profile
Laminated ruler paper (prepared in-house)	n/a	n/a
Methyl cellulose (viscosity: 400 cP)	Sigma	M0262
Photoshop software	Adobe	n/a	used for image processing
Rhodamine B isothiocyanate-Dextran	Sigma	r8881-100mg	"liquid" condition in the manuscript
Xenogen IVIS 200	Perkin Elmer	124262	In vivo imaging system