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 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.
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&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.
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
2. Anesthesia
3. Surgery
4. Gastrointestinal transit assay
5. Paraffin embedding and hematoxylin and eosin (HE) staining
6. Immune cell isolation and flow cytometry
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.
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 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.
The authors have nothing to disclose.
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&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).
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 |
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