Name: studying murine small bowel mechanosensing of luminal particulates
Uploaded: 2022-03-18T15:00:00
Description: Watch this Scientific Journal Video about Studying Murine Small Bowel Mechanosensing of Luminal Particulates at JoVE.com

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).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic.
1. Setup
<ol>
	<li>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.</li>
	<li>Cool 15 mL of distilled water in a 50 mL conical tube in a 4 &#176;C refrigerator.</li>
	<li>Heat anot...

<ol>
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E., Anderson, B., Bouchoucha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+movement+with+evaluating+colonic+transit+in+humans.">More movement with evaluating colonic transit in humans.</a> Neurogastroenterology and Motility. 31 (2), 13541 (2019).</li><li>Camilleri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gastric+emptying+and+colonic+filling+of+solids+characterized+by+a+new+method.">Human gastric emptying and colonic filling of solids characterized by a new method.</a> American Journal of Physiology. 257 (2), 284-290 (1989).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-illumination+intestine+projection+imaging+of+intestinal+motility+in+mice.">Trans-illumination intestine projection imaging of intestinal motility in mice.</a> Nature Communications. 12 (1), 1682 (2021).</li><li>Woting, A., Blaut, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestinal+permeability+and+gut-transit+time+determined+with+low+and+high+molecular+weight+fluorescein+isothiocyanate-dextrans+in+C3H+mice.">Small intestinal permeability and gut-transit time determined with low and high molecular weight fluorescein isothiocyanate-dextrans in C3H mice.</a> Nutrients. 10 (6), 685 (2018).</li><li>Miller, M. S., Galligan, J. J., Burks, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+measurement+of+intestinal+transit+in+the+rat.">Accurate measurement of intestinal transit in the rat.</a> The Journal of Pharmacologial and Toxicological Methods. 6 (3), 211-217 (1981).</li><li>Moore, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+carbon+monoxide+suppresses+the+development+of+postoperative+ileus+in+the+murine+small+intestine.">Inhaled carbon monoxide suppresses the development of postoperative ileus in the murine small intestine.</a> Gastroenterology. 124 (2), 377-391 (2003).</li><li>Machholz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments. (67), e2771 (2012).</li><li>Baeyens, N., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+vascular+mechanosensation+and+remodeling.">Biomechanics of vascular mechanosensation and remodeling.</a> Molecular Biology of the Cell. 27 (1), 7-11 (2016).</li><li>Ye, G. J., Nesmith, A. P., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanotransduction+on+vascular+smooth+muscle+myocytes'+cytoskeleton+and+contractile+function.">The role of mechanotransduction on vascular smooth muscle myocytes' cytoskeleton and contractile function.</a> The Anatomical Record (Hoboken). 297 (9), 1758-1769 (2014).</li><li>Mercado-Perez, A., Beyder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+feelings:+mechanosensing+in+the+gastrointestinal+tract.">Gut feelings: mechanosensing in the gastrointestinal tract.</a> Nature Reviews Gastroenterology &amp; Hepatology. , 1-14 (2022).</li><li>Brierley, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice.">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127 (1), 166-178 (2004).</li><li>Inoue, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet+and+abdominal+autofluorescence+detected+by+in+vivo+fluorescence+imaging+of+living+mice.">Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice.</a> Molecular Imaging. 7 (1), 21-27 (2008).</li><li>Szarka, L. A., Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+assessment+of+small-bowel+and+colonic+transit.">Methods for the assessment of small-bowel and colonic transit.</a> Seminars in Nuclear Medicine. 42 (2), 113-123 (2012).</li><li>Padmanabhan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+transit+measurements+in+mice+with+99mTc-DTPA-labeled+activated+charcoal+using+NanoSPECT-CT.">Gastrointestinal transit measurements in mice with 99mTc-DTPA-labeled activated charcoal using NanoSPECT-CT.</a> European Journal of Nuclear Medicine and Molecular Imaging. 3 (1), 1-8 (2013).</li><li>Jang, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+discrimination+in+rat+and+mouse+gastric+emptying.">Size discrimination in rat and mouse gastric emptying.</a> Biopharmaceutics and Drug Disposition. 34 (2), 107-124 (2013).</li><li>Zhu, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+sensory+neurons+communicate+with+interstitial+cells+of+Cajal+to+affect+pacemaker+activity+in+the+small+intestine.">Enteric sensory neurons communicate with interstitial cells of Cajal to affect pacemaker activity in the small intestine.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 446 (7), 1467-1475 (2014).</li><li>Treichel, A. 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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 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&#39;s primary function is to absorb substances critical for life. The small intestine is specifically responsible for nutrient absorption. The small intestine&#39;s transit is tightly regulated to match the digestion and absorption functions, resulting in various motility patterns. Bayliss and Starling described the &#34;law of the intestine&#34;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 (&#60;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td>other mice can be used with this protocol</td>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel software</td>
			<td>Microsoft</td>
			<td>n/a</td>
			<td>used for spreadsheet analysis</td>
		</tr>
		<tr>
			<td>Fluorescent Green Polyethylene Microspheres 1.00g/cc 75-90um - 10g</td>
			<td>Cospheric</td>
			<td>UVPMS-BG-1.00 75-90um - 10g</td>
			<td>&#34;smaller beads&#34; in the manuscript</td>
		</tr>
		<tr>
			<td>Fluorescent Green Polyethylene Microspheres 1.00g/cc 180-212um - 10g</td>
			<td>Cospheric</td>
			<td>UVPMS-BG-1.00 180-212um - 10g</td>
			<td>&#34;larger beads&#34; in the manuscript</td>
		</tr>
		<tr>
			<td>Gavage needles</td>
			<td>Instech</td>
			<td>FTP-18-50-50</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>used to extract fluorescence profile</td>
		</tr>
		<tr>
			<td>Laminated ruler paper (prepared in-house)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl cellulose (viscosity: 400 cP)</td>
			<td>Sigma</td>
			<td>M0262</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop software</td>
			<td>Adobe</td>
			<td>n/a</td>
			<td>used for image processing</td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate-Dextran</td>
			<td>Sigma</td>
			<td>r8881-100mg</td>
			<td>&#34;liquid&#34; condition in the manuscript</td>
		</tr>
		<tr>
			<td>Xenogen IVIS 200</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
	</tbody>
</table>

studying murine small bowel mechanosensing of luminal particulates

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.

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...

Watch this Scientific Journal Video about Studying Murine Small Bowel Mechanosensing of Luminal Particulates at JoVE.com

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

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 ...

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