We are grateful to the Laboratory Animal Center, Beijing Clinical Research Institute, and Beijing Friendship Hospital for providing animal care. This work was supported by the National Key Technologies R&#38;D Program (No. 2015BAI13B09), Beijing Natural Science Foundation (No. 7232035), National Natural Science Foundation of China (No. 82171823, 82374190), and Distinguished Young Scholars from Beijing Friendship Hospital (No. yyqcjh2022-4).

Animal care and experimental procedures were conducted in accordance with the Guiding Principles in the Care and Use of Animals (China) and were approved by the Ethics Review Committee of Beijing Friendship Hospital (NO. 20-2056). C57BL/6 mice (8-12 weeks old) were used for the study.
1. Preparation for surgery
<ol>
	<li>Fast all mice for 12 h prior to the planned modeling to meet the standardization of experimental conditions (Figure 1A).</li>
	<li>Prepare and sterilize surgical instruments.</li>
	<li>Prepare the anesthetic tribromoethanol and the heating pad. Monitor the heating pad to prevent overheating and keep it at a consistent temperature (37.5 &#176;C).</li>
</ol>
2. Anesthesia
<ol>
	<li>Anesthetize each mouse with tribromoethanol.
	<ol>
		<li>Dilute tribromoethanol to prepare a 20 mg/mL solution in normal saline. Administer 0.2 mL of the tribromoethanol working solution per 10 g of body weight via intraperitoneal injection. Use ophthalmic ointment on mice&#39;s eyes to prevent dryness while under anesthesia.</li>
		<li>Ensure mice are completely anesthetized within 5 min and remain anesthetized for 20 min.</li>
	</ol>
	</li>
	<li>Assess the depth of anesthesia by observing the inability of the mouse to remain upright and check for muscle relaxation. Observe the loss of voluntary movement, blink reflex, and response to reflex stimulation (toe or tail pinch with firm pressure).</li>
	<li>Assess the respiratory rate and pattern of the mouse by monitoring the movement of the chest wall and abdomen. Ensure the breathing rate is ~ 55-65 breaths/min under optimal anesthesia.</li>
	<li>Place the anesthetized mouse on a board and secure it with tape.</li>
	<li>After the mice were completely anesthetized, use an electric hair shaver to remove hair from the abdomen. After shaving, wipe off all loose hair on the abdomen using a saline-moistened cotton ball. 
	NOTE: Ensure proper ventilation and use of a fume hood or biosafety cabinet when handling tribromoethanol to minimize exposure risks. During the procedure, wear appropriate personal protective equipment, including gloves and lab coats, to prevent accidental contact with chemicals and biological materials.</li>
</ol>
3. Surgery
<ol>
	<li>Fully extend the limbs to expose the abdomen. Ensure that the mouse&#39;s head is positioned to maintain a clear airway (Figure 1B).</li>
	<li>Disinfect the skin of the surgical area twice using a cotton ball soaked with 75% alcohol. After disinfection, use a dry, sterile medical gauze to remove excess alcohol from the abdomen. Administer lidocaine (10 mg/kg, subcutaneously) at the intended site prior to the incision.</li>
	<li>Use a sterile scalpel to make an incision in the skin and lift the rectus abdominis muscle in the middle of the abdomen using tweezers.
	<ol>
		<li>Make a small incision along the median line of the rectus abdominis muscle, taking care to avoid injury to various organs in the abdomen.</li>
		<li>Ensure the approximate range of the incision is as follows. Ensure that the upper margin of the incision is 6-8 mm from the xiphoid process of the sternum, the lower margin is 6-8 mm from the external genitalia, and the length of the incision is ~1 cm.</li>
	</ol>
	</li>
	<li>Place a piece of sterile surgical drape or sterile gauze&#160;on both sides of the abdominal incision. Use hemostatic forceps to fix the sterile gauze on the upper and lower edges of the incision, exposing the incision (Figure 1C). 
	NOTE: In the sham group, the incision was covered with wet gauze for 5 min without any surgical operation.</li>
	<li>Properly fix the sterile drape or gauze, then use two cotton swabs pre-moistened with saline to gently press against both sides of the abdominal wall adjacent to the incision. Squeeze out a small amount of the intestinal tube through the incision and expose it by placing it on the sterile drape or gauze (Figure 1D).</li>
	<li>Completely moisten two sterile cotton swabs with normal saline. Gently grasp the intestinal tissue with the moistened cotton swabs and carefully remove the small intestine.
	<ol>
		<li>Locate the caecum and then take out the intestine with the sterile cotton swabs until 2 cm before the stomach to avoid touching the pancreas. Extend the intestine from the proximal end of the pancreato-duodenal ligament to the distal end of the ileocecal region.</li>
	</ol>
	</li>
	<li>Apply consistent pressure along the entire small intestine from the proximal to the distal ends. Ensure uniform force application for 5 min until small bleeding spots emerge on the intestinal surface (Figure 1E).</li>
	<li>After 5 min, use pre-moistened sterile cotton swabs to carefully place all the small intestines back into the abdominal cavity, following the normal physiological anatomical position of the small intestine.</li>
	<li>Gently massage the abdomen across the sterile gauze and abdominal wall for 3-5 s to ensure that the bowel is restored to its natural anatomical position and to prevent artificial mechanical intestinal obstruction or mesenteric torsion after surgery.</li>
	<li>Inject 100 &#181;L of saline into the abdominal cavity to replace lost fluids during the operation and lubricate the abdominal tissue.</li>
	<li>Close the abdomen using 6-0 surgical suture and perform a two-layer closure at the abdominal incision. Begin by closing the muscle layer with continuous sutures, taking care to avoid damage to the abdominal organs. Lift the rectus abdominis muscle during suturing (Figure 1F).</li>
	<li>After completely closing the rectus abdominis muscle, suture it completely with simple intermittent sutures using a 6-0 surgical suture. Maintain a stitch length of ~0.5 mm and a needle spacing of ~2 mm.</li>
	<li>After closing the abdominal incision, gently wipe the area near the incision with dry sterile medical gauze to keep it dry and clean from blood, tissue fluid, or normal saline.</li>
	<li>Apply a small amount of medical incision adhesive to and around the incision to avoid splitting after surgery.</li>
	<li>After the medical incision adhesive has completely dried, transfer the mice to a paper towel, and place the cage paper towel on a heated blanket maintained at a constant temperature of 37.5 &#176;C until the mice fully recover. Do not leave the animals unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Transfer the awake mice to the animal feeding barrier environment and allow them to drink freely until the detection time.</li>
</ol>
4. Gastrointestinal transit assay
<ol>
	<li>Administrate 200 &#181;L of FITC-dextran solution (5 mg/mL in PBS) to the fasted mice via gavage 22.5 h after surgery (Figure 1A).</li>
	<li>Euthanize the mice using a method approved by the Institutional Animal Care and Use Committee (IACUC), such as CO2 inhalation followed by cervical dislocation.
	<ol>
		<li>To isolate the digestive tract, make a midline incision from the diaphragm to the pelvis, carefully removing the surrounding connective tissues and organs.</li>
		<li>Separate the digestive tract from the surrounding tissues and organs, ensuring that the entire tract from the stomach to the anus is included.</li>
	</ol>
	</li>
	<li>Divide the digestive tract into 15 equal segments (from stomach to colon), numbered sequentially from 1 to 15 based on physiological position (sequence: St -S1:S10-Ce-L1:L3) (Figure 2A).</li>
	<li>Cut each segment of intestinal tissue with its content into pieces measuring 1-4 mm and place them into separate 1.5 mL centrifuge tubes containing 1 mL of DPBS.</li>
	<li>Vortex the centrifuge tubes for 10 s to homogenize the samples.</li>
	<li>Centrifuge the tubes at 500 &#215; g for 1 min. Collect the supernatant for FITC fluorescence quantification using a multimode microplate reader.</li>
	<li>Describe the gastrointestinal transit by calculating the geometric center (GC) of the FITC-dextran and calculate using the following formula: (&#931;[% FITC per segment x segment number])/100.</li>
</ol>
5. Paraffin embedding and hematoxylin and eosin (HE) staining
<ol>
	<li>Euthanize the mice by CO2 inhalation followed by cervical dislocation after 24 h. Perform paraffin embedding and gastrointestinal assays on the same animal.</li>
	<li>Open the abdominal cavity and collect small intestinal tissue for further analysis.</li>
	<li>Fix the entire bowel with intestinal content in Carnoy&#39;s fixative (60% methanol + 10% acetic acid + 30% chloroform) at 4 &#176;C for 2 h.</li>
	<li>Deacidify the tissue by rinsing twice with methanol for 30 min each time.</li>
	<li>Replace methanol with ethanol twice for 30 min each time.</li>
	<li>Deacidify the tissue by rinsing twice with methanol for 30 min each time. To achieve tissue transparency with xylene, immerse the tissue in xylene and incubate for 1 h.</li>
	<li>Embed the tissue in paraffin wax using a paraffin embedding machine. Cut the embedded tissue into 4 &#181;m thick slices using a microtome. 
	NOTE: Divide the small intestine into different waxes for improved performance. Properly label the segments to identify their location.</li>
	<li>Perform HE staining using the HE staining kit. Seal the slides and examine them under a microscope for analysis.</li>
</ol>
6. Immune cell isolation and flow cytometry
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Predigestion solution: Prepare Hank&#39;s balanced salt solution without Ca2+ and Mg2+ with 1 mM dithiothreitol (DTT) and 10 mM ethylenediaminetetraacetic acid (EDTA).</li>
		<li>Digestion solution: Prepare a solution containing 200 &#181;g/mL DNAase I, 500 &#181;g/mL collagenase IV, 4% FBS, and 100 &#181;M HEPES buffer in RPMI 1640.</li>
		<li>Magnetic-activated cell sorting works (MACS) buffer: Prepare a buffer containing 5 g/L BSA and 2 mM EDTA in PBS without Ca2+ or Mg2+.</li>
	</ol>
	</li>
	<li>Remove fecal contents by washing the intestines with normal saline. Remove Peyer&#39;s patches and connective tissue of the small intestine. Cut the small intestines open longitudinally and then cut into 1 cm segments in ice-cold PBS.</li>
	<li>Wash the intestine segments with predigestion solution at 37 &#176;C for 20 min on an orbital shaker. Collect the predigestion solution and filter it through 70 &#181;m cell strainers to obtain intraepithelial lymphocytes (IELs).</li>
	<li>Digest the segments in the digestion solution for 30 min and then filter the suspension through 70 &#181;m cell strainers to obtain lamina propria lymphocytes (LPLs).</li>
	<li>Centrifuge the LPL cells at 500 &#215; g for 10 min, and discard the supernatant.</li>
	<li>Wash and suspend the cells with DPBS (sodium azide, Tris, and protein-free) at a concentration of 1 x 106 cells/100 &#181;L.</li>
	<li>Incubate the cells with a fluorescent viability dye (1:500) for 15 min at room temperature (RT) in the dark.</li>
	<li>Suspend the cells with MACS buffer and centrifuge them at 500 &#215; g for 5 min.</li>
	<li>Count the stained cells using a hemocytometer or automated cell counter. Resuspend the cells at a concentration of 1 x 106 cells/100 &#181;L of MACS buffer. Perform antibody staining (1:200-1:400) by adding the appropriate volume of antibody to the cell suspension and incubating at 4 &#176;C for 15 min.</li>
	<li>Wash the cells as described in step 6.8.</li>
	<li>Detect the samples using flow cytometry and analyze data with the associated flow cytometry software7,8.</li>
</ol>

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

<ol>
	<li>Chapman, S. J., Pericleous, A., Downey, C., Jayne, D. G. <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+following+major+colorectal+surgery.">Postoperative ileus following major colorectal surgery.</a> Br J Surg. 105 (7), 797-810 (2018).</li><li>Farro, G., 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=CCR2-dependent+monocyte-derived+macrophages+resolve+inflammation+and+restore+gut+motility+in+postoperative+ileus.">CCR2-dependent monocyte-derived macrophages resolve inflammation and restore gut motility in postoperative ileus.</a> Gut. 66 (12), 2098-2109 (2017).</li><li>Wouters, M. M., Vicario, M., Santos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+functional+GI+disorders.">The role of mast cells in functional GI disorders.</a> Gut. 65 (1), 155-168 (2016).</li><li>Engel, D. R., 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=T+helper+type+1+memory+cells+disseminate+postoperative+ileus+over+the+entire+intestinal+tract.">T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract.</a> Nat Med. 16 (12), 1407-1413 (2010).</li><li>Schwartz, C., Voehringer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+murine+basophils+by+flow+cytometry+and+histology.">Identification of murine basophils by flow cytometry and histology.</a> Methods Mol Biol. 2163, 367-375 (2020).</li><li>Funk, E. K., Weissbrod, P., Horgan, S., Orosco, R. K., Califano, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+experience+with+a+novel+single-port+platform+for+transoral+surgery.">Preclinical experience with a novel single-port platform for transoral surgery.</a> Surg Endosc. 35 (8), 4857-4864 (2021).</li><li>Nemoto, S., Mailloux, A. W., Kroeger, J., Mul&#233;, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMIP-031:+Immunologic+checkpoint+expression+on+murine+effector+and+memory+T-cell+subsets.">OMIP-031: Immunologic checkpoint expression on murine effector and memory T-cell subsets.</a> Cytometry A. 89 (5), 427-429 (2016).</li><li>Brandi, J., Wiethe, C., Riehn, M., Jacobs, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMIP-93:+A+41-color+high+parameter+panel+to+characterize+various+co-inhibitory+molecules+and+their+ligands+in+the+lymphoid+and+myeloid+compartment+in+mice.">OMIP-93: A 41-color high parameter panel to characterize various co-inhibitory molecules and their ligands in the lymphoid and myeloid compartment in mice.</a> Cytometry A. 103 (8), 624-630 (2023).</li><li>Swidsinski, A., Weber, J., Loening-Baucke, V., Hale, L. P., Lochs, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+organization+and+composition+of+the+mucosal+flora+in+patients+with+inflammatory+bowel+disease.">Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease.</a> J Clin Microbiol. 43 (7), 3380-3389 (2005).</li><li>Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O., Yang, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Swiss-rolling+technique+for+intestinal+tissue+preparation+for+immunohistochemical+and+immunofluorescent+analyses.">Improved Swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses.</a> J Vis Exp. (113), e54161 (2016).</li><li>van Bree, S. H., 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=Novel+method+for+studying+postoperative+ileus+in+mice.">Novel method for studying postoperative ileus in mice.</a> Int J Physiol Pathophysiol Pharmacol. 4 (4), 219-227 (2012).</li></ol>

The authors have nothing to disclose.

The success of surgery relies on several critical steps. First, maintaining consistency during intestinal intramural (IM) surgery is imperative to induce extensive injury to the small intestine. Proper pressure applied during the IM procedure and the resulting hyperemic effect on the intestine are crucial for surgical success. The observation of the entire digestive tract turning pink and displaying red hemorrhagic spots after rubbing with a cotton swab served as an indicator of a successful operation. Additionally, ensu...

Postoperative ileus (POI) is a syndrome that poses a significant challenge in the field of human health, particularly in the management of patients undergoing abdominal surgery. Characterized by delayed recovery of gastrointestinal motility, POI contributes to prolonged hospital stays and increased health care costs, yet no established definition, etiology, or treatment exists1. Recent research has shed light on the pivotal role of immune cells in the progression of POI2,3,4, yet further investigation is required to elucidate the underlying mechanisms involved.
In this protocol, we introduce a murine model of POI induced by intra-abdominal surgery, which closely mimics the impact of abdominal surgery on the digestive tract. Our goal was to provide a standardized method for modeling POI in mice, enabling researchers to investigate its pathophysiology and explore novel therapeutic interventions.
The rationale behind the development and utilization of this technique lies in the need for reliable preclinical models to study POI. Traditional approaches to studying POI often lack translational relevance or fail to capture the complex interplay of factors contributing to the condition. By introducing a murine model that closely replicates the clinical scenario, researchers can more accurately investigate the mechanisms underlying POI and test potential therapeutic interventions in a controlled experimental setting.
Compared to alternative techniques, the murine model of POI presented in this protocol offers several advantages. Initially, we integrated our experimental findings with recent advancements to establish a standardized and reproducible protocol for inducing POI in experimental animals. This protocol facilitates consistent assessment of gastrointestinal transit function. Second, employing histological staining and flow cytometry enabled the assessment of tissue swelling, immune cell proliferation, and activation, yielding valuable insights into the inflammatory processes underlying POI5.
In the broader context of the literature, establishing a murine model of POI contributes to the expanding body of research aimed at comprehending the pathophysiology of this condition. By bridging the gap between basic science and clinical practice, preclinical models play a pivotal role in developing novel therapeutic strategies for POI6. Moreover, the availability of standardized animal models enhances the reproducibility and comparability of research findings across different laboratories. However, this POI model relies on mechanical stimulation during the surgical procedure. Other forms of stimulation-induced ileus may not be suitable for this model. Additionally, researchers should consider factors such as animal welfare regulations, ethical considerations, and resource availability when planning experiments using this model.
In summary, the introduction of a murine model of POI signifies a noteworthy advancement in preclinical research on this debilitating condition. Additionally, we employed H&#38;E staining and flow cytometry to assess tissue swelling and immune cell proliferation and activation. The establishment of a murine POI model would facilitate the discovery of POI mechanisms and promote the development of novel therapies for POI.

<table><tbody><tr><td>1 M HEPES</td><td>Thermo</td><td>15630080</td><td></td></tr><tr><td>APC anti-mouse I-A/I-E (MHC-II)</td><td>Biolegend</td><td>107614</td><td></td></tr><tr><td>APC anti-mouse TCRb</td><td>Biolegend</td><td>109212</td><td></td></tr><tr><td>APC/Cy7 anti-mouse CD4</td><td>Biolegend</td><td>100414</td><td></td></tr><tr><td>APC/Cy7 anti-mouse Ly6G</td><td>Biolegend</td><td>127624</td><td></td></tr><tr><td>Brilliant Violet 421 anti-mouse CD69</td><td>Biolegend</td><td>104545</td><td></td></tr><tr><td>Brilliant Violet 421 anti-mouse F4/80</td><td>Biolegend</td><td>123132</td><td></td></tr><tr><td>Brilliant Violet 785 anti-mouse/human CD44</td><td>Biolegend</td><td>103041</td><td></td></tr><tr><td>BUV395 anti-mouse CD8a</td><td>BD</td><td>563786</td><td></td></tr><tr><td>BUV737 anti-mouse CD3e</td><td>BD</td><td>612771</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Culture Microscope</td><td>CKX53</td><td>Olympus</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas (&lt;strong&gt;DNase I&lt;/strong&gt;)</td><td>Sigma-Aldrich</td><td>DN25-5G</td><td></td></tr><tr><td>DL-Dithiothreitol solution</td><td>Sigma-Aldrich</td><td>43816-10ML</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS-100G</td><td></td></tr><tr><td>FITC anti-mouse CD45</td><td>Biolegend</td><td>147709</td><td></td></tr><tr><td>FITC-dextran (70 kWM)</td><td>Sigma-Aldrich</td><td>FD70-100MG</td><td>Gastrointestinal Transit Assay</td></tr><tr><td>HE staining kit</td><td>solarbio</td><td>G1120</td><td></td></tr><tr><td>PE anti-mouse CD11b</td><td>Biolegend</td><td>101208</td><td></td></tr><tr><td>PE anti-mouse PD-1</td><td>Biolegend</td><td>114118</td><td></td></tr><tr><td>PE/Cy7 anti-mouse CD11c</td><td>Biolegend</td><td>117318</td><td></td></tr><tr><td>Percoll</td><td>GE (Pharmacia)</td><td>17-0891-01</td><td></td></tr><tr><td>Symphony A5 Flow cytometer</td><td>BD</td><td>-</td><td>Immune cell detection and sorting</td></tr><tr><td>Tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402</td><td>Anesthesia</td></tr><tr><td>Varioskan LUX</td><td>Thermo</td><td>N16699</td><td>Multimode microplate reader</td></tr><tr><td>Zombie Aqua Fixable Viability kit</td><td>Biolegend</td><td>423102</td><td>Fluorescent viability dye&amp;nbsp;</td></tr></tbody></table>

postoperative ileus murine model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

In this protocol, POI was surgically induced by intestinal manipulation (IM), which is similar to the effect of clinical surgery. In the sham group, an incision was made without the IM. POI mice were sacrificed 24 h post-POI surgery along with sham control mice. The critical function of the digestive tract, content transit function, was detected by gavage of FITC-dextran. The POI model was considered successful because the FITC intensity increased in the proximal part of the small intestine&#160;(Fig...

Postoperative Ileus Murine Model

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Research

JoVE Journal

Immunology and Infection

496 Views.  Capital Medical University. De reikwijdte van onze studie is intestinale immuniteit. We proberen de pathofysiologie en het mechanisme van POI-letsels te illustreren voor de ontwikkeling van nieuwe therapieën. Een uitdaging is het opzetten van consistente en reproduceerbare experimentele modellen van POI in muizen.Dit omvat het beheersen van de variabelen, zoals chirurgische technieken, en het beoordelen van protocollen in de dierenverzorging om de variabiliteit tussen experiment...

Video: Postoperatief Ileus muizenmodel

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.

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 ...

Medicine

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Cancer Research

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 ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

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, ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Biology

Murine Ileal Resection Model: Studying Ileectomy-Induced Bile Metabolism in Murine Model

Source: Zhang, R., et al. <a href="https://www.jove.com/v/55728"> Ileectomy-induced Bile Overaccumulation in Mouse Intestine.</a> J. Vis. Exp. (2017)
This video describes the detailed protocol for intestinal resection in a murine model. This technique provides insights into ileectomy-induced bile malabsorption, overaccumulation, and toxicity in the mouse intestine.

Source: Zhang, R., et al.  Ileectomy-induced Bile Overaccumulation in Mouse Intestine. J. Vis. Exp. (2017)
This video describes the detailed protocol for ...

Postoperative Ileus Murine Model

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