Name: Postoperative Ileus Murine Model (Video) | JoVE Journal
Uploaded: 2024-07-12T13:10:00.000+00:00
Duration: 4 min 26 s
Description: Learn to establish a reproducible murine model of postoperative ileus through intestinal manipulation. This method enables researchers to study intestinal immunity and evaluate novel anti-inflammatory therapies.

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5 minutes

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/pubmed?term=((Postoperative+ileus+following+major+colorectal+surgery%5BTitle%5D)+AND+%22Br+J+Surg%22%5BJournal%5D)">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/pubmed?term=((CCR2-dependent+monocyte-derived+macrophages+resolve+inflammation+and+restore+gut+motility+in+postoperative+ileus%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">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/pubmed?term=((The+role+of+mast+cells+in+functional+GI+disorders%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">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/pubmed?term=((T+helper+type+1+memory+cells+disseminate+postoperative+ileus+over+the+entire+intestinal+tract%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((Identification+of+murine+basophils+by+flow+cytometry+and+histology%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Preclinical+experience+with+a+novel+single-port+platform+for+transoral+surgery%5BTitle%5D)+AND+%22Surg+Endosc%22%5BJournal%5D)">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., Mulé, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OMIP-031%3A+Immunologic+checkpoint+expression+on+murine+effector+and+memory+T-cell+subsets%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">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/pubmed?term=((OMIP-93%3A+A+41-color+high+parameter+panel+to+characterize+various+co-inhibitory+molecules+and+their+ligands+in+the+lymphoid+and+myeloid+compartment+in+mice%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">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/pubmed?term=((Spatial+organization+and+composition+of+the+mucosal+flora+in+patients+with+inflammatory+bowel+disease%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Improved+Swiss-rolling+technique+for+intestinal+tissue+preparation+for+immunohistochemical+and+immunofluorescent+analyses%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Novel+method+for+studying+postoperative+ileus+in+mice%5BTitle%5D)+AND+%22Int+J+Physiol+Pathophysiol+Pharmacol%22%5BJournal%5D)">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, ensuring that the mean GC value remains at approximately 5 after surgery indicates that the model was successful compared to that of sham-operated mice. Microscopic examination of tissue swelling in the submucosa and immune cell infiltration, as demonstrated by HE staining, also serves as a critical step in validating the severity of POI.
Modifications to the protocol may be necessary to optimize outcomes or troubleshoot any challenges encountered. For instance, to accurately display morphological changes in the intestinal epithelium, embedding the intestinal tissue along with its contents using Carnoy&#39;s fixative is recommended9. This unique embedding protocol ensures quick fixation, which is essential for preserving tissue morphology. However, if the investigator&#39;s objectives focus on the epithelium or lamina propria, employing the improved Swiss rolling technique for wax embedding of intestinal tissue preparation is advisable10. This technique compresses the interspace of intestinal tissue and ensures a consistent direction of villi, facilitating the embedding of the entire small intestine in one wax-embedded sample.
Despite its utility, the protocol has limitations. One limitation is the reliance on animal models, which may not fully replicate the complexity of human pathophysiology. Additionally, variations in surgical technique and individual mouse responses may introduce variability in outcomes. According to our recent study, using a consistent operator is an important principle for reducing operator-dependent variability. Moreover, Van et al. introduced a self-made device capable of applying a specific weight to the intestine, ensuring consistent pressure on the IM across different mice11. This approach indeed proves valuable in maintaining the consistency of the model.
This protocol represents a valuable contribution to the field by providing a standardized method for inducing POI through IM surgery in mice. By elucidating critical steps and offering modifications for tissue embedding and immune cell isolation, this protocol enhances reproducibility and reliability in POI modeling. Utilizing flow cytometry7,8 for immune cell analysis allows for a comprehensive investigation of the immune response underlying POI pathogenesis, complementing existing techniques and expanding our understanding of this complex syndrome.
The protocol presented here holds promise for future preclinical research focused on understanding and treating POI. By refining surgical techniques and optimizing tissue processing methods, researchers can further explore the mechanisms underlying POI development and identify novel therapeutic targets. Additionally, the compatibility of the protocol with flow cytometry enables in-depth immune profiling, opening avenues for investigating immune-modulating interventions aimed at alleviating POI symptoms. Overall, the protocol lays the groundwork for advancing our knowledge of POI and developing targeted therapeutic strategies to improve patient outcomes.

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

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 (Figure 2B). The mean GCs were calculated to quantify the transit dysfunction oftheIM and indicate the consistency of the operation (Figure 2C).
HE staining indicated the morphology of different segments of the digestive tract (Figure 3). The submucosa and smooth muscle layer of the duodenum and ileum became swollen 24 h after POI induction (Figure 3A,B). Compared to those in sham mice, HE staining of the duodenum revealed increased immune cell infiltration in POI mice (Figure 3A).
Multicolor flow cytometric analysis is the central method for analyzing the proportion, status, and function of immune cells. The intestinal tissue was digested with collagenase and deoxyribonuclease and separated into a single-cell suspension for flow cytometry. The gating strategy was used to determine the proportions of CD3 T cells and related subpopulations among the LPLs (Figure 4A). The activation and adhesion marker CD69 and the expression of CD4 T cells, CD8 T cells, and gd T cells are upregulated in POI mice (Figure 4B)7. Similarly, the proportion of the CD44+ PD-1+ (activation, terminal differentiation, and exhaustion marker) subpopulation showed an increasing tendency (Figure 4C)7. In addition to lymphocytes, the proportions of myeloid cell subsets (neutrophils, dendritic cells, and macrophages) are also increased after IM surgery (Figure 4D)8.
<img alt="Mouse surgery timeline, fasting duration, FITC-Dex gavage experiment, preclinical research steps." class="xfigimg" src="/files/ftp_upload/66465/66465fig1v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Murine model of postoperative ileus. (A) Schematic of the murine model of postoperative ileus. (B) Mouse fixation after anesthesia and shaving. (C) The incision was exposed and covered with a medical sterile gauze. (D) Intestinal tubes were squeezed out of the abdominal cavity through the abdominal incision and exposed by placing them on sterile gauze. (E) The whole small intestine was exposed to a moistened sterile cotton swab. (F) The muscle layer was closed by surgical suture. <a href="https://www.jove.com/files/ftp_upload/66465/66465fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Intestinal FITC distribution; panel A shows sections, panel B graphs intensity, panel C compares centers." class="xfigimg" src="/files/ftp_upload/66465/66465fig02v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Gastrointestinal transit assay for digestive tract function assessment. At 22.5 h after surgery, the sham or POI mice were gavaged with FITC-dextran solution and sacrificed 90 min later. (A) The digestive tract was divided into 15 segments and numbered according to physiologic position. (B) FITC intensity of every segment from the digestive tracts of sham and POI mice. (C) The geometric centers of the administered FITC-dextran were calculated and are shown as violin plots. <a href="https://www.jove.com/files/ftp_upload/66465/66465fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histology comparison of duodenum and ileum tissues, sham vs POI, 100μm and 25μm magnified views." class="xfigimg" src="/files/ftp_upload/66465/66465fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Representative HE staining of the small intestine of POI mice. HE staining of cross-sections of the (A) duodenum and (B) ileum from sham and POI mice. Scale bars: 100 µm or 25 µm (zoomed-in image of the inset). <a href="https://www.jove.com/files/ftp_upload/66465/66465fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow cytometry analysis diagrams: CD4, CD8, gd T cells, CD45+ cells identification, immune response study." class="xfigimg" src="/files/ftp_upload/66465/66465fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Representative flow cytometric analysis of small intestinal immune cells from POI mice. (A) Flow cytometry gating strategy for T cell populations from small intestinal tissues of POI mice 24 h after surgery. (B) The proportions of T cell active marker CD69+ in CD4 T cells, CD8 T cells, and gd T cells from the small intestine of sham and POI mice. (C) The proportions of CD44+ PD-1+ active markers among CD4 T cells, CD8 T cells, and gd T cells in the small intestines of sham and POI mice. (D) The proportions of neutrophils (Ly6G+ CD11b+), dendritic cells (CD11c+ MHC-II+), and macrophages (CD11b+ F4/80+) among CD45+ Aqua- live immune cells from the small intestines of sham and POI mice. The number in each panel represents the percentage of the respective cell types. <a href="https://www.jove.com/files/ftp_upload/66465/66465fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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

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.

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

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Murine Ileocolic Bowel Resection with Primary Anastomosis

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A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

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.

Murine Model of Intestinal Ischemia-reperfusion Injury

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.

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

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.

A Mouse Model of Intestinal Partial Obstruction

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

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

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.

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

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.

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

Learn to establish a reproducible murine model of postoperative ileus through intestinal manipulation. This method enables researchers to study intestinal immunity and evaluate novel anti-inflammatory therapies.

Postoperative Ileus Murine Model

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