To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.
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.
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 known today as the peristaltic reflex; the segment proximal to the food bolus contracts to propel it forward, and the distal segment relaxes to receive it. In theory, this pattern alone could be sufficient to transport material aborally, but over a century of research has painted a more complex picture of contractile activity in the GI tract. Three small intestine motility periods are recognized in humans: the migrating motor complex (MMC), the fasting period, and the postprandial period3. The same patterns have been reported in mice4,5. The MMC is a cyclic motor pattern conserved across most mammals6,7. The MMC has a characteristic four-phase pattern that serves as a useful clinical marker in functional GI disorders7. The four phases, in order of occurrence, are (I) quiescence, (II) irregular, low amplitude contractions, (III) regular high amplitude contractions, and (IV) ramp-down period of declining activity7. The MMC marks the major motor pattern of the fasting period3. MMCs of the fasting period clear up the contents of the small intestine in preparation for the next meal.
The motor patterns of the postprandial period are optimized for the digestive and absorptive functions3. Regardless of caloric composition, initial transit is quick along the small intestine, contents are spread along the length of the bowel, and transit subsequently slows down8. Absorption is optimized by increasing contact surface area and slowing it down to increase residence time. Once the nutrients are inside the lumen, the dominant pattern consists of close (<2 cm apart) uncoordinated contractions (segmenting contractions), with a few superimposed large-amplitude contractions spanning the whole length of the small bowel (peristaltic contractions)9. Segmenting contractions mix the intraluminal contents in place. The occasional large peristaltic contractions propel the contents towards the colon.
The timing of this transition back to MMCs depends on food volume and caloric composition10. Thus, the small bowel samples luminal cues to regulate when to transition between motility periods. Mechanical cues, such as physical properties of luminal contents11, luminal volume, and wall tension, engage mechanoreceptor cells in the GI wall12,13,14,15,16. Indeed, increasing the solid component of a meal leads to an increase in small bowel transit17. We speculate that physical properties, such as the liquid or solid state of intraluminal contents, must engage different mechanoreceptors due to the various forces they generate on the GI wall18.
The gold standard for measuring in vivo GI transit in humans, as in mice, is the use of radioactive tracers measured by scintigraphy as they exit the stomach or transit along the colon19,20. In mammals, the small bowel loops in unpredictable ways making the small bowel difficult to image in vivo reliably, but progress is being made21. Further, there is currently a lack of tools to quantify how the small bowel handles particulates of varying properties and sizes. The starting point here was a gold-standard technique that standardizes the study of small bowel transit22,23,24 and barrier function22. It consists of gavaging mice with a fluorescent material, waiting for GI motility to transport the material, excising the GI tract, segmenting it into several sections from stomach to colon, sectioning, and homogenizing intraluminal contents for fluorescence quantification. We made two improvements. First, we altered the makeup of the gavaged contents to include fluorescent microscopic beads to determine how the small bowel distributes physical particulates. Second, we improved the spatial resolution by imaging the whole GI tract from stomach to colon ex vivo and used variable size binning to standardize our analysis across animals. We postulate that this reveals novel insights into the balance of propulsive versus segmenting contractions during the postprandial phase.
The GI tract, like other tubular organs, such as blood vessels, requires mechanical sensors and effectors to maintain homeostasis26,27,28. 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…
The authors have nothing to disclose.
We thank Mrs. Lyndsay Busby for administrative assistance and Mr. Joel Pino for media support. NIH grants supported this work: DK123549, AT010875, DK052766, DK128913, and Mayo Clinic Center for Cell Signaling in Gastroenterology (DK084567).
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 |