This work was supported by grants from the Deutsche Forschungsgemeinschaft (KA1270/3-1/2) and BONFOR (O-112.0040).

1. Animals
Male C57Bl6/J mice with a mean weight of 25 g were obtained from Harlan Winkelmann (Borchen, Germany). All experiments were performed in accordance with the federal law regarding the protection of animals. The principles of laboratory animal care were followed. Animals were maintained on a 12-hr light/dark cycle and provided with commercially available rodent chow and tap water ad libitum. Animals should be housed at least 7 days after arrival in your animal facility before experiments. Note that all experiments can be easily adapted to rats or other species and with slight modifications into large animal models.
2. Operative Procedure
<ol>
	<li>Anesthesia is induced and maintained with isoflurane (Abbott, Wiesbaden, Germany) in oxygen. After onset of anesthesia place animals in supine position and fix extremities with adhesive tape (Leukosilk, Beiersdorf, Hamburg, Germany).</li>
	<li>After shaving and surgical disinfection of the abdominal wall a median laparotomy is performed on a length of 2 centimeters. Enter the abdominal cavity via an incision along the Linea alba. Place two sterile retractors to keep the abdomen open. Carefully eventerate the small bowel to the left and place it on a wet cotton gauze.</li>
	<li>Evacuate the entire small bowel luminal contents with two moist and sterile cotton applicators (MaiMed, Neunkirchen, Germany) by simultaneously rolling the applicator from the pylorus to the cecum. CAUTION: Avoid hemorrhages of the small bowel wall or lesions of the gut vessels. Put the gut back into the peritoneal cavern without twisting to prevent ischemia or mechanical obstruction and close the abdominal incision using a two layer continuous 5/0 polyamid non-absorbable sutures (Ethicon, Norderstedt, Germany).</li>
</ol>
3. Postoperative Care
<ol>
	<li>After the procedure place the animal under a heating lamp, observing it until it recovered from anesthesia. Put the mouse back in its cage and allow access to water and food. Due to the property of opioids to affect peristalsis, we perform perioperative analgesia with NSAIDs (for example metamizol) in drinking water.</li>
</ol>
4. Functional Studies 24 hr after IM
<ol class="lalpha">
	<li>Gastrointestinal Transit (GIT)
	<ol>
		<li>Anaesthetize mice slightly with isoflurane in oxygen 22.5 hr after IM. Carefully hyperextend the head of the mouse and pull out the tongue with a blunt forceps. Fill a 1 ml syringe with at least 100 &mu;l fluorescein-labeled dextran (FITC-dextran, 70,000 MW, Sigma Aldrich, Taufkirchen, Germany), connect it to an arterial catheter (Vygon, Ecouen, France) and carefully insert the catheter as a gastric tube into the stomach.</li>
		<li>Briefly inject 100 &mu;l FITC-dextran and pull out the catheter. Let the animal awake and wait for 90 min. During this time mice have access to food or water.</li>
		<li>Prepare fifteen 2 ml spinning tubes and label them consecutively.</li>
		<li>Euthanize mice 90 min after gavage and remove the entire gastrointestinal tract from stomach to rectum. Place the intestine onto a polystyrene pad and avoid stretching. CAVE: It is crucial to remove the gut gently to avoid shifting of liquid FITC-dextrane into the nearby segment. For the beginning it might be helpful to use clips at the beginning and at the end of each segment.</li>
		<li>Measure full length of small bowel and full length of colon. Divide the small bowel into 10 equal sized segments by pinning it down to the polystyrene pad with cannulas. Divide the colon into 3 segments. You have prepared 15 segments (stomach #1), ten equally sized small bowel segments (#2-11), cecum (#12) and three 3 equally sized colonic segments (#13-15).</li>
		<li>Transect the intestine at the marked positions, grab it with a forceps and flush them once with 1.0 ml KHB into the previously prepared KHB containing 2.0 ml tubes and vortex vigorously for 20 sec. The stomach and cecum are placed directly and cut in the prepared tubes. Fill these tubes with 1.0 ml KHB, thereby rinsing traces of luminal contents from the scissor into the tubes.</li>
		<li>Centrifuge the probes at 12,000 rcf for 5 min at room temperature in a table top centrifuge. During centrifugation prepare another 15 consecutively numbered 1.5 ml tubes.</li>
		<li>Transfer the clear supernatants into the new 1.5 ml tube and store in the dark at 4 &deg;C until analysis (next step). The probes can be stored for several days at 4 &deg;C without significant loss of FITC signal. For longer storage add 0.09% Natrium-azide or store at &lt; -18 &deg;C.</li>
		<li>Pipette 100 &mu;l duplets of the supernatants on a black 96-well plate (Greiner Bio One, Frickenhausen, Germany). Pipette two 100 &mu;l samples of KHB as blank.</li>
		<li>Read the plate in a fluorescence reader (Safire, Tecan, Crailsheim, Germany) and quantify the fluorescence at 494 nm (absorption) / 521 nm (emission) wavelength. Subtract blank values from samples.</li>
		<li>Calculate the geometric center (GC) of FITC-dextran distribution, which is actually the center of gravity for the distribution of marker, by the following formula: GC = &sum; (% of total fluorescent signal per segment * segment number) / 100</li>
	</ol>
	 Note: By multiplying percentage of fluorescence of each segment with its segment number a weighted mean of the distribution of marker within the intestine is assessed. This point is influenced by both the distribution and distance traveled by marker, but assumes no specific underlying distribution.7 The calculated GC value is often presented in combination with a graph demonstrating the distribution of FITC dextran over the gastrointestinal tract (Figure 1A). </li>
	<li>Colonic Transit
	<ol>
		<li>Mice should be weakly anaesthetized with isoflurane 2 % in oxygen. CAUTION: Ensure that the animal will awake within 40 sec.</li>
		<li>Carefully examine colonic patency by inserting a fistula probe (metal rod, 2 mm diameter) through the anus into the colon.</li>
		<li>Using the fistula probe push forward a 2 mm glass ball 3 cm into the colon and pull out the probe immediately. Place the mouse in a transparent cage and start measuring the time after the fistula probe was pulled out of the colon. Keep an eye to the mouse and stop the time directly after the glass ball became excreted.</li>
	</ol>
	</li>
</ol>
5. Histochemical Analysis of Isolated ME Specimens
<ol>
	<li>Midjejunal segments are cut from the bowel and immersed in cold KHB in a Sylgard-filled dish.</li>
	<li>Mesentery is pinned to the dish with 0.2 mm insect needles (Fine Science Tools, Heidelberg, Germany).</li>
	<li>Cut open the segment along its mesenteric border and stretch to ~120 % of its length and width. Finalize the fixation with insect pins on opposite site of the mesentery. Rinse the sylgard dish carefully with fresh ice cold KHB, thereby flushing all luminal contents. 
	ME can now mechanically separated from the tunica mucosa starting from the mesentery. During preparation replace fresh chilled KHB several times.</li>
	<li>Separated tunica mucosa can be shock frozen in liquid nitrogen for further analysis or discarded.</li>
	<li>Fix the isolated ME whole mount with 100 % ethanol for 10 min at room temperature. 
	Wash the whole mount three times using chilled KHB. Note: other fixatives (i.e. 4% formaldehyde, acetic acid, aceton) can also be used after testing its compatibility with the subsequent analysis.</li>
	<li>The ME whole mounts are ready for further histochemical or immunhistochemical staining:</li>
</ol>
<ol class="lalpha">
	<li>Histochemistry: myeloperoxidase (MPO) staining 
	Infiltrated PMNs can be examined by the following MPO staining protocol.
	<ol>
		<li>Solve 10 mg Hanker Yates reagent (Polyscience Europe, Eppelheim, Germany) in 10 ml KHB and add 100 &mu;l of 3% H2O2. Prepare the solution freshly immediately before use and do not reuse.</li>
		<li>Incubate whole mounts for 10 min at room temperature.</li>
		<li>Rinse specimens extensively with cold KHB and incubate for 10 min.</li>
		<li>Mount specimens in aqueous mounting medium (Aquatek, Merck, Darmstadt, Germany) After drying for 2 hr analyze slides at under a light microscope.</li>
		<li>Count numbers of cells in 5 randomly chosen fields and calculate as MPO positive cells / mm2.</li>
	</ol>
	</li>
	<li>Immunohistochemical Staining: 
	Accordingly to the histochemical MPO staining, immunohistochemical stainings of the whole mounts can be performed. Principally, your established immunohistochemical staining protocols of cell cultures or tissue slices can be easily adapted to a whole mount staining. However, optimizing the procedure for specific antibodies is strongly recommend.
	<ol>
		<li>Cut specimens into 5 x 5 mm pieces and perform staining in 2.0 ml round bottom centrifuge tubes in ~150 &mu;l - 200 &mu;l antibody solution.</li>
		<li>Fixation and blocking procedure (i.e. 3% BSA in PBS for 1 hr at room temperature) can be performed in the "sylgard dish".</li>
		<li>Washing steps in PBS or other buffers can be performed in 12-well plates.</li>
		<li>Transfer the .specimens carefully with a sharp forceps between staining tubes and washing plates.</li>
		<li>After the final washing procedure mount specimens in anti-fading mounting medium with cover slips and analyze with a fluorescent microscope.</li>
	</ol>
	</li>
</ol>
6. Organ Culture of ME
<ol>
	<li>The whole small intestine is resected 24 hr after IM and placed in cold KHB containing 200 U/ml penicillin G and 100 &mu;g/ml streptomycin (KHB + P/S).</li>
	<li>Cut the gut into 3 cm lengths and pin each segment down to a Sylgard containing glass dish.</li>
	<li>Remove the mesentery with fine scissors and slip the intestine on a knitting needle.</li>
	<li>Gently incise the ME with a sharp forceps and strip it off from the submucosa using a moist cotton applicator. The tunica mucosa remains on the knitting needle and can be snap frozen for further investigation. The ME is collected in cold KHB + P/S.</li>
	<li>Cut the isolated ME strips into small pieces of 2 - 4 mm length and incubate it in ice cold KHB + P/S for half an hour and rinse the specimen several times.</li>
	<li>Transfer approximately 50-60 mg ME (at least half ME of one small bowel) in a sterile 24-well plate containing 500 &mu;l Dulbecco s modified Eagle medium (DMEM).</li>
	<li>Incubate at 37 &deg;C and 5 % CO2 for additional 24 hr.</li>
	<li>Inspect the cell cultures for bacterial or fungal contamination under a microscope.</li>
	<li>Collect supernatant, spin down for 5 min at 1,000 rcf and snap freeze in liquid nitrogen. Meanwhile blot dry the muscle tissue on a clean tissue for 30 sec and weight.</li>
	<li>Supernatants can be used for subsequent analysis of released cytokines (IL-6) by ELISA or other metabolites (i.e. nitrite) in the supernatant by following manufactures instructions.</li>
	<li>Normalize measured concentrations per weight of ME tissue.</li>
</ol>
7. Representative Results
<ol>
	<li>Functional Studies 
	The most important parameter to evaluate the severity of POI is the examination of GIT in vivo. Figure 1A demonstrates a typical distribution of FITC dextran along the gastrointestinal tract in untreated controls and intestinally manipulated mice 24 hr after surgery. "Sham operated" animals showed a normal GIT with a calculated geometric center (GC) in the cecum while IM led to a significant delay of GIT in the proximal jejunum (Figure 1B) 
	Colonic motility was separately focused on by measurement of the ball excretion time of a 2 mm glass ball. Sham operated mice show an excretion time of 48 - 200 sec while IM led to a prolonged excretion time between 470 - 775 sec (Figure 1C).</li>
	<li>Morphological Studies 
	To describe the inflammatory extend within the ME several phenotypes of leukocytes could be analyzed using histochemistry or immunohistochemistry. In figure 2A+B a low presence of PMNs in "sham operated" animals was observed (39&plusmn;7 cells/mm2). IM of the small bowel led to a strong PMN infiltration (660&plusmn;86 cells/mm2) compared with sham operated mice</li>
	<li>ME Organ Culture 
	Many inflammatory mediators liberated from cells are difficult to detect in tissue lysates. Organ cultures allow detection of liberated mediators in the culture supernatant. A representative mediator in POI is NO, which is as major inhibitory neurotransmitter in the gastrointestinal tract.5 
	NO could not be detected in ME organ culture supernatants of unoperated control animals (5&plusmn;6 &mu;M/ 24 hr /mg tissue). In sham operated (laparotomy) mice basal NO levels of 53&plusmn;36 &mu;M/ 24 hr /mg tissue were observed. ME cultures of intestinally manipulated (harvested 24 hr after surgery) mice produce significantly more NO (2254&plusmn;853 &mu;M/ 24 hr /mg tissue) during 24 hr ME organ culture (Figure 3).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/4086/4086fig1.jpg" /> 
Figure 1. Effect of IM on GIT (A+B) and colonic transit (C) GIT was measured as the percent of non-absorbable fluorescein-labeled dextran in 15 gastrointestinal segments-stomach (Sto), small intestine (SI 1-9), cecum (Cec), and colon (Co)-90 min after oral ingestion. Colonic transit was measured as the time from pulling out the fistula probe until excretion of a 2 mm glass bead. (A)Gastrointestinal distribution of fluorescein isothiocyanate- dextran after sham operation or IM. In sham operated animals most of the marker is located in the cecum or the proximal colon compared with proximal jejunal location in manipulated animals. Dotted lines indicate calculated GC. (B) Calculation of the GC demonstrated a prolonged gastrointestinal transit time after IM. (C) Colonic transit time showed a significant delay in IM mice compared with sham operated animals. GC for the 15 intestinal segments are displayed as mean (n=5). Colonic transit times were displayed mean with all individual values (n=6). *** = p&lt;0.001, Student's t-test.
<img alt="Figure 2" src="/files/ftp_upload/4086/4086fig2.jpg" /> 
Figure 2. Detection of MPO positive cells within small bowel ME.(A) ME whole mounts were stained with Hanker Yates reagent for detection of MPO positive PMN 24 hr after laparotomy (sham) or IM procedure. (B) Quantification of MPO positive cell within 5 randomly chosen fields per mouse. n= 6 animals per group. *** = p&lt;0.001, Student's t-test.
<img alt="Figure 3" src="/files/ftp_upload/4086/4086fig3.jpg" /> 
Figure 3. NO production in culture supernatants of ME. Muscle specimens from unoperated controls, sham operated and IM mice were taken 24 hr postoperatively and cultured for 24 hr. Untreated mice showed only a very low basal production of NO. After laparotomy NO liberation is increased without measuring significant differences between the control group and sham operated mice. 24 hr after IM NO production is dramatically increased compared with untreated or sham operated mice. n= 5 animals per group. *** = p&lt;0.001, 1 way ANOVA.

<ol>
	<li>Wehner, S., Behrendt, F. F., Lyutenski, B. N., Lysson, M., Bauer, A. J., Hirner, A., Kalff, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+macrophage+function+prevents+intestinal+inflammation+and+postoperative+ileus+in+rodents.">Inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents.</a> Gut. 56, 176-185 (2007).</li><li>Kalff, J. C., Schwarz, N. T., Walgenbach, K. J., Schraut, W. H., Bauer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocytes+of+the+intestinal+muscularis:+their+phenotype+and+isolation.">Leukocytes of the intestinal muscularis: their phenotype and isolation.</a> J. Leukoc. Biol. 63, 683-691 (1998).</li><li>Kehlet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+ileus.">Postoperative ileus.</a> Gut. 47, iv85-iv86 (2000).</li><li>Wehner, S., Schwarz, N. T., Hundsdoerfer, R., Hierholzer, C., Tweardy, D. J., Billiar, T. R., Bauer, A. J., Kalff, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IL-6+within+the+rodent+intestinal+muscularis+after+intestinal+surgical+stress.">Induction of IL-6 within the rodent intestinal muscularis after intestinal surgical stress.</a> Surgery. 137, 436-446 (2005).</li><li>Kalff, J. C., Schraut, W. H., Billiar, T. R., Simmons, R. L., Bauer, A. 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No conflicts of interest declared.

A comparative study analyzing different levels of IM (small bowel eventration for 10 min versus gently inspection of the small bowel using two cotton applicators versus IM with evacuation of small bowel content into the cecum) revealed a correlation between the extent of surgical manipulation and POI. Compared with the other groups IM showed the highest amount of leukocytes in the ME (PNMs, macrophages and mast cells) resulting in prolonged and severe POI.8.
When establishing the mo...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Ethilon 5/0</td>
 <td>Ethicon</td>
 <td>1666H</td>
 </tr>
 <tr>
 <td>Cotton applicators</td>
 <td>MaiMed</td>
 <td>71010</td>
 </tr>
 <tr>
 <td>arterial catheter</td>
 <td>Vygon</td>
 <td>115-798</td>
 </tr>
 <tr>
 <td>96 well plate (black)</td>
 <td>Greiner Bio One</td>
 <td>655096</td>
 </tr>
 <tr>
 <td>glass ball (2 mm diameter)</td>
 <td>Worf</td>
 <td>available on request</td>
 </tr>
 <tr>
 <td>Insect pins</td>
 <td>Fine Science Tools</td>
 <td>26000-25</td>
 </tr>
 <tr>
 <td>Hanker Yates Reagent</td>
 <td>Polyscience</td>
 <td>560694</td>
 </tr>
 <tr>
 <td>Aquatek</td>
 <td>Merck</td>
 <td>HC109850</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Lonza</td>
 <td>BE12-604 F</td>
 </tr>
 <tr>
 <td>FITC-dextran (MW 70000)</td>
 <td>Sigma Aldrich</td>
 <td>46945-500MG-F</td>
 </tr>
 </tbody>
</table>

functional assessment of intestinal motility and gut wall inflammation in rodents: analyses in a standardized model of intestinal manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Watch this Scientific Journal Video about Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation at JoVE.com

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

functional-assessment-intestinal-motility-gut-wall-inflammation

Research

JoVE Journal

Medicine

22.0K vues.  University of Bonn. Iléus postopératoire (POI) est une complication de la chirurgie abdominale conduisant à une morbidité accrue et un séjour prolongé à l'hôpital. Parce que les stratégies prophylactiques ou thérapeutiques font défaut intensification de la recherche est nécessaire. Par conséquent, nous avons établi un modèle de souris standard et possible d'étudier la physiopathologie de POI et d'étudier les options possibles thérapeutiques.

Vidéo: Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...