Postoperative Ileus Murine Model

Summary

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.

Abstract

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.

Introduction

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 POI^2,3,4, 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&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.

Protocol

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

1. Fast all mice for 12 h prior to the planned modeling to prevent aspiration death caused by reflux of stomach contents after anesthesia (Figure 1A).
2. Prepare and sterilize surgical instruments.
3. Prepare the anesthetic tribromoethanol and the heating pad. Monitor the heating pad to prevent overheating and keep it at a consistent temperature (37.5 °C).

2. Anesthesia

1. Anesthetize each mouse with tribromoethanol.
   1. 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.
   2. Ensure mice are completely anesthetized within 5 min and remain anesthetized for 20 min.
2. 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).
3. 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.
4. Place the anesthetized mouse on a board and secure it with tape.
5. 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.

3. Surgery

1. Fully extend the limbs to expose the abdomen. Ensure that the mouse's head is positioned to maintain a clear airway (Figure 1B).
2. 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.
3. Make an incision through the skin and lift the rectus abdominis muscle in the middle of the abdomen using tweezers.
   1. Make a small incision along the median line of the rectus abdominis muscle, taking care to avoid injury to various organs in the abdomen.
   2. 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.
4. Place a piece of gauze pre-moistened with normal saline on both sides of the abdominal incision. Use hemostatic forceps to fix the 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.
5. Properly fix the 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 gauze (Figure 1D).
6. Completely moisten two cotton swabs with normal saline. Gently grasp the intestinal tissue with the moistened cotton swabs and carefully remove the small intestine.
   1. Locate the caecum and then take out the intestine with the 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.
7. 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).
8. After 5 min, use pre-moistened cotton swabs to carefully place all the small intestines back into the abdominal cavity, following the normal physiological anatomical position of the small intestine.
9. Gently massage the abdomen across the 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.
10. Inject 100 µL of saline into the abdominal cavity to replace lost fluids during the operation and lubricate the abdominal tissue.
11. Remove the gauze and close the abdomen using 6-0 surgical sutures. 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).
12. 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.
13. 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.
14. Apply a small amount of medical incision adhesive to and around the incision to avoid splitting after surgery.
15. After the medical incision adhesive has completely dried, transfer the mice to a cage equipped with dry, cleaning bedding, and place the cage on a heated blanket maintained at a constant temperature of 37.5 °C until the mice fully recover. Do not leave the animals unattended until they have regained sufficient consciousness to maintain sternal recumbency.
16. Transfer the awake mice to the animal feeding barrier environment and allow them to drink freely until the detection time.

4. Gastrointestinal transit assay

1. Administrate 200 µL of FITC-dextran solution (5 mg/mL in PBS) to the fasted mice via gavage 22.5 h after surgery (Figure 1A).
2. Euthanize the mice using a method approved by the Institutional Animal Care and Use Committee (IACUC), such as CO₂ inhalation followed by cervical dislocation.
   1. To isolate the digestive tract, make a midline incision from the diaphragm to the pelvis, carefully removing the surrounding connective tissues and organs.
   2. Separate the digestive tract from the surrounding tissues and organs, ensuring that the entire tract from the stomach to the anus is included.
3. 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).
4. 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.
5. Vortex the centrifuge tubes for 10 s to homogenize the samples.
6. Centrifuge the tubes at 500 × g for 1 min. Collect the supernatant for FITC fluorescence quantification using a multimode microplate reader.
7. Describe the gastrointestinal transit by calculating the geometric center (GC) of the FITC-dextran and calculate using the following formula: (Σ[% FITC per segment x segment number])/100.

5. Paraffin embedding and hematoxylin and eosin (HE) staining

1. Euthanize the mice by CO₂ inhalation followed by cervical dislocation after 24 h. Perform paraffin embedding and gastrointestinal assays on the same animal.
2. Open the abdominal cavity and collect small intestinal tissue for further analysis.
3. Fix the entire bowel with intestinal content in Carnoy's fixative (60% methanol + 10% acetic acid + 30% chloroform) at 4 °C for 2 h.
4. Deacidify the tissue by rinsing twice with methanol for 30 min each time.
5. Replace methanol with ethanol twice for 30 min each time.
6. 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.
7. Embed the tissue in paraffin wax using a paraffin embedding machine. Cut the embedded tissue into 4 µm thick slices using a microtome.
   NOTE: Divide the small intestine into different waxes for improved performance. Properly label the segments to identify their location.
8. Perform HE staining using the HE staining kit. Seal the slides and examine them under a microscope for analysis.

6. Immune cell isolation and flow cytometry

1. Prepare the following solutions.
   1. Predigestion solution: Prepare Hank's balanced salt solution without Ca²⁺ and Mg²⁺ with 1 mM dithiothreitol (DTT) and 10 mM ethylenediaminetetraacetic acid (EDTA).
   2. Digestion solution: Prepare a solution containing 200 µg/mL DNAase I, 500 µg/mL collagenase IV, 4% FBS, and 100 µM HEPES buffer in RPMI 1640.
   3. 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⁺.
2. Remove fecal contents by washing the intestines with normal saline. Remove Peyer'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.
3. Wash the intestine segments with predigestion solution at 37 °C for 20 min on an orbital shaker. Collect the predigestion solution and filter it through 70 µm cell strainers to obtain intraepithelial lymphocytes (IELs).
4. Digest the segments in the digestion solution for 30 min and then filter the suspension through 70 µm cell strainers to obtain lamina propria lymphocytes (LPLs).
5. Centrifuge the LPL cells at 500 × g for 10 min, and discard the supernatant.
6. Wash and suspend the cells with DPBS (sodium azide, Tris, and protein-free) at a concentration of 1 x 10⁶ cells/100 µL.
7. Incubate the cells with a fluorescent viability dye (1:500) for 15 min at room temperature (RT) in the dark.
8. Suspend the cells with MACS buffer and centrifuge them at 500 × g for 5 min.
9. Count the stained cells using a hemocytometer or automated cell counter. Resuspend the cells at a concentration of 1 x 10⁶ cells/100 µL 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 °C for 15 min.
10. Wash the cells as described in step 6.8.
11. Detect the samples using flow cytometry and analyze data with the associated flow cytometry software^7,8.

Representative Results

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.

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 sterile medical gauze. (D) Intestinal tubes were squeezed out of the abdominal cavity through the abdominal incision and exposed by placing them on gauze. (E) The whole small intestine was exposed to a moistened cotton swab. (F) The muscle layer was closed by surgical suture. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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). Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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's fixative is recommended⁹. This unique embedding protocol ensures quick fixation, which is essential for preserving tissue morphology. However, if the investigator'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 cytometry^7,8 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.

Materials

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