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 another 15 mL of distilled water in a beaker using a hot plate with a magnetic stir bar to approximately 80-90 &#176;C.</li>
	<li>Measure 0.5% methylcellulose for a total of 30 mL (0.15 g).</li>
	<li>Gently add methylcellulose to 15 mL of hot water so that it does not clump. The methylcellulose solution will be cloudy during this process.</li>
	<li>Once dissolved, remove the beaker from the hot plate and add the 15 mL of chilled water to the hot beaker.</li>
	<li>Place in a 4 &#176;C fridge until the solution is clear, approximately 15-20 min.</li>
	<li>When clear, weigh 3 mg of rhodamine isothiocyanate (RITC)-dextran per 5 mL of 0.5% methylcellulose solution and mix to combine. This is the liquid condition in the Representative Results section.
	<ol>
		<li>Alternatively, weigh 25 mg of fluorescent polyethylene microspheres and mix with 200 &#181;L of the methylcellulose solution until well incorporated. Thorough incorporation of microspheres is ensured by vortex prior to gavage. The experimenter must visualize homogeneous distribution of suspended microspheres in the mixture.</li>
		<li>As the RITC-dextran solution is light sensitive, refrigerate the solution. Prepare the RITC-dextran solution and the microshpere mixture in advance and use within 2 months. Resuspend the microsphere mixture before use. 
		&#8203;NOTE: Microspheres of varying sizes are used to study gastrointestinal handling of different materials. In the Representative Results section, we demonstrate the results of using small (diameter: 75-90 &#181;m) and larger (diameter: 180-212 &#181;m) microspheres.</li>
	</ol>
	</li>
</ol>
2. Intraluminal content gavage
<ol>
	<li>Prepare the gavage by attaching an 18 Ga x 50 mm feeding tube to a 1 mL syringe and drawing 200 &#181;L of RITC solution or microsphere solution.</li>
	<li>Manually restrain the fasted animal using the one-handed restraint technique25. Refer to the institution&#39;s information on proper murine restraint techniques.</li>
	<li>Gently insert the feeding tube through the mouth and esophagus of the mouse until it enters the stomach. 
	&#8203;NOTE: The gavage step is critical for a successful experiment. It requires experienced hands that can consistently insert the tube roughly the same distance in each mouse. This step needs to be standardized by experimenters carrying out the protocol. The same experimenter should gavage all mice cohorts that will be compared to each other.</li>
	<li>Slowly expel the syringe contents into the stomach and carefully remove the tube from the mouse.</li>
	<li>Following the gavage, return the mouse to the cage.</li>
	<li>Dispose of the gavage tube.</li>
	<li>Repeat the process as needed for the desired number of animals.</li>
</ol>
3. Bowel dissection
<ol>
	<li>Before beginning the dissections, turn on the in vivo imaging instrument to allow it to come to temperature.</li>
	<li>Sacrifice the mouse 30 min after the gavage. Use carbon dioxide inhalation to sacrifice the mouse, followed by cervical dislocation to ensure successful euthanasia.</li>
	<li>After confirming successful euthanasia, place the mouse in supine position on a dissection stage and pin its four appendages on the stage to have access to the abdomen. Wet the surface of the abdomen with 70% ethanol to wet the abdominal hairs.</li>
	<li>Use micro-dissection forceps (#5) to pull the skin and acquire sharp surgical scissors to make a transverse incision 1 cm above the anus. To expose the intraperitoneal cavity, continue the incision vertically up the abdomen until the rib cage.</li>
	<li>Gently move the cecum to the left with the micro-dissection forceps to expose the distal colon.</li>
	<li>Cut the distal colon with micro-dissection scissors just proximal to the rectum.</li>
	<li>Gently unravel the colon, cecum, and small intestine by pulling slowly in the opposite direction. 
	NOTE: Maintaining the dissected bowels in one continuous segment will create the most ease for the investigator. Rips and tears along the segment will cause subsequent steps to be more difficult.</li>
	<li>Use micro-dissection scissors to cut proximal to the stomach.</li>
	<li>Use forceps to transfer the dissected bowels to the measurement sheet (Figure 1). Place the stomach at 0 mm and arrange the bowels along the ruler until 200 mm. Use micro-dissection scissors to cut at 200 mm. Repeat this process of aligning the bowels from 0 mm to 200 mm along the rulers while remaining confident that the orientation of the bowels does not get confused.
	<ol>
		<li>Be careful to avoid stretching the bowels while aligning to the rulers.</li>
	</ol>
	</li>
	<li>Once the tissue is arranged on the ruler, arrange the cecum so that it is parallel with the tissue but not in direct contact with it (if any fluorescence is found within that region, then a clear difference distinction between the cecum and the intestines will be needed).</li>
	<li>Place the measurement sheet with the dissected tissue in a dark area so the fluorescence can be preserved until it is time to image.</li>
</ol>
4. Ex vivo imaging
<ol>
	<li>Open the in vivo imaging software and log in.</li>
	<li>Initialize the imaging instrument so it is ready for image acquisition.</li>
	<li>Set the &#39;Excitation&#39; and &#39;Emission&#39; fields to the corresponding color used for the bead or RITC gavage. Red (liquid): excitation 535 nm /emission 600 nm. Green (microspheres): excitation 465 nm /emission 520 nm.</li>
	<li>Set the exposure to &#39;Auto&#39;.</li>
	<li>Select the field of view.</li>
	<li>Before placing the measurement sheet into the instrument, ensure the intestines have not shifted during transport.</li>
	<li>Place the measurement sheet into the instrument within the field of view.</li>
	<li>Securely shut the door to the instrument and select Snapshot to photograph the field of view.</li>
	<li>Save collected pictures to a flash drive for analysis. Save individual captures of the fluorescent and photograph images. An overlay of the photograph and fluorescence indicates the location of fluorescent material in the GI tract (Figure 1). 
	&#8203;NOTE: We recommend saving the files in the Tag Image File (.tif) format for downstream processing.</li>
</ol>
5. Analysis
<ol>
	<li>Open the fluorescent and photograph image files in a picture editing software.</li>
	<li>Adjust the pixel size of both images such that they have the exact same dimensions (our convention was to set both images to 1280 pixels by 850 pixels [height x width]).</li>
	<li>Close the photograph file. The following steps involve only the fluorescent image.</li>
	<li>Use an eraser tool to remove the background and make it transparent. An eraser may be needed to remove patches of the remaining background.</li>
	<li>Create a new layer. Make it an entirely black background to the fluorescent signal. This can be achieved by choosing a black fill to the layer and dragging the layer to lie below the layer with the fluorescent image.</li>
	<li>Save the new fluorescent image, containing only the fluorescent signal on a black background, as a new .tif file.</li>
	<li>Open the new fluorescent and photograph images in ImageJ.</li>
	<li>Turn each image into a 32-bit image by choosing Image &#62; Type &#62; 32-bit.</li>
	<li>Create a merged image of both by choosing Image &#62; Color &#62; Merge Channels. In the dialog box that opens, select the photograph file for the gray channel and the fluorescent file under any colored channels.</li>
	<li>Turn off the scale of the merged image by selecting Analyze &#62; Set Scale &#62; Click to Remove Scale.</li>
	<li>Select the &#34;Rectangle&#34; tool in ImageJ.</li>
	<li>Draw a rectangle around a section of the small bowel. Pay close attention to the width of this region of interest (ROI), as it should remain constant between all ROIs. The nominal value is not essential, just that it is kept consistent across all ROIs drawn on this image [since the small bowel takes over several rows, individual ROIs will be drawn over each section of the small bowel in a different row (Figure 1)]. Figure 2 shows the location of the width value in the ImageJ toolbar.</li>
	<li>Duplicate the ROI by selecting Image &#62; Duplicate. Select only the channel corresponding to the colored channel.</li>
	<li>Use the rectangle tool again to draw an ROI over the whole new image.</li>
	<li>Retrieve a profile of the fluorescence by selecting Analyze &#62; Plot Profile. The resulting graph plots on the y-axis the average intensity for each pixel along the length of the ROI. This was selected in step 5.12 to be the length of the analyzed section of the small bowel</li>
	<li>Open the list of values and copy them to a spreadsheet software.</li>
	<li>Repeat steps 5.12-5.16 for each section of the small bowel on a different ruler row. Keep pasting the values on the same spreadsheet file immediately under each previous set of values, such that all continuous rows in that column contain the average intensity value for the entire length of the small bowel.</li>
	<li>In the spreadsheet software, multiply each average intensity value by the constant width of the ROI rectangle created in step 5.12. This will yield the real intensity value along the small bowel at each point. 
	NOTE: Different animals will have slight variations on small bowel length. To prevent the length differences from affecting the outcome of downstream data analysis, the string of intensity values needs to be binned into a consistent number of bins across all experimental samples (Figure 3, Figure 4, and Figure 5 display results for three bin sizes).</li>
	<li>Divide the number of intensity values by the number of bins desired. The resulting quotient S determines the number of intensity values being included in each bin.</li>
	<li>Determine the bin where each raw intensity value will go by using a roundup formula. This step places each raw intensity value into a bin. Each raw intensity value is chronologically indexed with the integer N. The quotient N/S determines the bin number assigned to each raw value. The roundup formula should round this quotient to a whole number with no decimals.</li>
	<li>Generate the value for each bin by using an averageif formula. The objective is to average the raw values assigned to a specific bin in Step 5.20. Hence, the arguments in the formula should be: (1) assigned bins generated in Step 5.20, (2) bin number, (3) raw intensity values. 
	NOTE: The first analysis we can perform on the binned data is a geometric center analysis, which quantifies how far we observe the highest fluorescent intensity along the small intestine (Figure 4).</li>
	<li>Normalize each intensity value over the total fluorescent intensity. In other words, divide each intensity value by the sum of all intensities.</li>
	<li>Multiply each normalized value by the bin number. The product reflects the relative weight of each bin, contributing to the total fluorescent intensity.</li>
	<li>Adding all values generated in Step 5.23 yields the bin number at the center of the fluorescent signal. Divide by the number of bins to express the geometric center as the fraction of small bowel traveled by the fluorescent center. 
	NOTE: To reflect the spatial distribution of the signal, the next step is to generate the power spectrum of the binned intensity data set (Figure 5).</li>
	<li>Open a Fast Fourier Transform (FFT) wizard in the spreadsheet software. Select the input as the binned data set and the output as an empty set of the same number of rows as bins.</li>
	<li>Extract the real value component of the FFT.</li>
	<li>Raise each real value of the FFT to the second power. This yields a data set of power spectra.</li>
	<li>Select the first half of the power spectra data set and move it so that it lies below the second half. This has the effect of centering the power spectra around the center frequency when it is plotted in the next step.</li>
	<li>Plot the power spectra on the y-axis of an x-y graph where the x-axis values are the range from -1 x (number of bins/2) to (number of bins/2) -1. In the case of 1000 bins, the axis values would range from -500 to 499.</li>
	<li>Power spectra for each animal should be compared on the basis of the spread of non-zero peaks and the heights of these non-zero peaks.</li>
</ol>

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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 ...

studying-murine-small-bowel-mechanosensing-of-luminal-particulates

Research

JoVE Journal

Medicine

1.8K Views.  Mayo Clinic. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Video: Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Murine Ileocolic Bowel Resection with Primary Anastomosis

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn&#8217;s disease and colon cancer being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease.

Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease.

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn&#8217;s disease and colon cancer ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder.
In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. ...

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Validating Xanthatin-Keap1 Interaction with Docking and CETSA

Protein Target Prediction and Validation of Small Molecule Compound

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of crucial protein targets have become integral to drug development. Molecular docking is a computational tool widely utilized to investigate protein-ligand binding, especially in the context of drug and protein target interactions. For the experimental verification of the binding and to access the binding of the drug and its target directly, the cellular thermal shift assay (CETSA) method is used. This study aimed to integrate molecular docking with CETSA to predict and validate interactions between drugs and vital protein targets. Specifically, we predicted the interaction between xanthatin and Keap1 protein as well as its binding mode through molecular docking analysis, followed by verification of the interaction using the CETSA assay. Our results demonstrated that xanthatin could establish hydrogen bonds with specific amino acid residues of Keap1 protein and reduce the thermostability of Keap1 protein, indicating that xanthatin could directly interact with Keap1 protein.

Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA

The experiment used here shows a method of molecular docking combined with cellular thermal shift assay to predict and validate the interaction between small molecules and protein targets.

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of ...