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 ....

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 meet the standardization of experimental conditions (Figure 1A).
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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, ensu.......

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