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. 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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 ...

This is a protocol that studies how the small intestine handles materials of different physical properties and how it distributes those materials in space. It's a complementary tool in our study of gut mechanosensitivity. The main advantages of this protocol are that it can be used to study materials or mixtures with different physical properties and that the resolution allows us to resolve regional transits over short intestinal segments.This technique adds to our understanding of gut mechanosensitivity. It helps us understand how the small intestine makes transit decisions based on the mechanical cues it receives from the intraluminal contents. To begin, prepare the gavage by attaching a feeding tube of 18-gauge thickness and 50-millimeters length to a 1-milliliter syringe and drawing 200 microliters of RITC solution or microsphere solution.Next, manually restrain the fasted animal using the one-handed restraint technique, then gently insert the feeding tube through the mouth and esophagus of the mouse until it enters the stomach. Once inserted, slowly expel the syringe contents into the stomach and carefully remove the tube from the mouse. Following the gavage, return the mouse to the cage.Before beginning the dissections, turn on the in vivo imaging instrument so it reaches the desired temperature. After euthanasia, place the mouse in the supine position on a dissection stage and pin its four appendages on the stage to access the abdomen. Next, wet the surface of the abdomen with 70%ethanol.Next, use micro-dissection forceps to pull the skin and make a transverse incision using sharp surgical scissors 1 centimeter above the anus. To expose the intraperitoneal cavity, continue the incision vertically up the abdomen until the rib cage. Gently handle the bowels and appreciate their orientation in the abdominal cavity before manipulating them.Cut the proximal to the gastroesophageal junction, thus separating the stomach from the esophagus and gently unravel the colon, cecum, and small intestine by pulling slowly in the opposite direction. Use micro-dissection scissors to separate the attachments of the mesentery to the bowels. Later, using the forceps, transfer the dissected bowels to the measurement sheet.Place the stomach at 0 millimeters and arrange the bowels along the ruler until 200 millimeters, then cut the bowel at 200 millimeters with micro-dissection scissors and repeat this procedure of bowel alignment. Once the tissue is arranged on the ruler, arrange the cecum to parallel with the tissue, but not in direct contact with it. Place the measurement sheet with the dissected tissue in a dark area so the fluorescence can be preserved until it is time to image.To start ex vivo imaging, open the imaging software. Log in and initialize the imaging instrument to be ready for image acquisition. For RITC gavage, set Excitation at 535 nanometers and Emissions, 600 nanometers.While for green microsphere beads, set Excitation at 465 nanometers and Emission at 520 nanometers. Now, set the exposure to Auto and select the field of view. After ensuring that the intestines have not shifted during transport, place the measurement sheet into the instrument within the field of view.Securely shut the door to the instrument and select Snapshot to photograph the field of view. Save collected pictures to a flash drive for analysis. Next, save individual captures of the fluorescent and photograph images.An overlay of the photograph and fluorescence indicates the location of fluorescent materials in the gastrointestinal tract. Open the fluorescent and photograph image files in picture editing software for analysis. Adjust the pixel size of both images to have the exact dimensions and close the photograph file.For the fluorescent image, use an eraser tool to remove the background and make it transparent. Create a new layer. Choose a black fill to the layer and drag the layer to lie below the layer with the fluorescent image and make an entirely black background.Save the new fluorescent image containing only the fluorescent signal on a black background as a new TIF file. Open the new fluorescent and photograph images in ImageJ. Turn each image into a 30-bit image by choosing Image, followed by Type and 30-bit.Create a merged image of both by choosing Image, followed by Color and Merge Channels. In the dialogue box that opens, select the photograph file for the gray channel and the fluorescent file Under any colored channels. Turn off the scale of the merged image by selecting Analyze, followed by Set Scale and Click to Remove Scale.Select the Rectangle tool in ImageJ. Draw a rectangle around a section of the small bowel while paying close attention to the width of this region of interest, as it should remain constant between all regions of interest. Duplicate the region of interest by selecting Image, followed by Duplicate and select only the channel corresponding to the colored channel.Again, use the Rectangle tool to draw a region of interest over the whole new image and retrieve a fluorescence profile by selecting Analyze, followed by Plot Profile. Open the list of values and copy them to spreadsheet software. Repeat steps from drawing of the region of interest to retrieving a fluorescence profile for each section of the small bowel as described previously on a different ruler row.In the spreadsheet software, multiply each average intensity value by the region of interest rectangle's constant width created in the previous step. This will yield the real intensity value along the small bowel at each point. Shown here are the average fluorescence traces along the length of the small bowel.The distribution of fluorescent material varies according to the material properties of the intraluminal contents. Increasing the number of bins used to sort the same raw data sets reveals granular trace features unresolvable with fewer bins. Smaller bins decrease measurement uncertainty, increase spatial resolution, and better reflect the distributive component of small bowel motility.The geometric center fails to completely characterize the spatial distribution of intraluminal contents. This limitation of the geometric center measurement is evident in the comparison between liquids and larger beads. The fluorescent trace of the larger beads is more distributed, but it averages out to a similar point as that of the liquid, regardless of binning granularity, which highlights the limitation of solely focusing on the geometric center.To account for the distributive nature of small bowel contractions, a power spectral analysis was incorporated in the present study. Plotting the power spectrum demonstrates that as bin sizes shrink, we can observe significantly dominant frequencies in the larger beads spectrum, but not in that of the small beads. Some, but not all, of those additional dominant frequencies are present in the liquid spectrum.The most delicate steps of this protocol are the gavage and the dissection. These should be standardized by the experimenters prior to performing this technique for experimental purposes. This is a terminal experiment, as the animal needs to be sacrificed.When possible, maximize the use of your animal model by performing other non-terminal experiments before this one. Our group has recently used this technique to support our finding of gut touch sets where the gut uses epithelial mechanoreceptors to sense finer physical properties, much like our fingers do.

Research

JoVE Journal

Medicine

Protocol for Long Duration Whole Body Hyperthermia in Mice

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