This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett’s esophagus and esophageal adenocarcinoma.
Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett’s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.
Gastroesophageal reflux disease (GERD) is a chronic disorder caused by the prolonged exposure of distal esophagus to gastric or gastroduodenal contents1. Prolonged exposure to these noxious refluxates impairs the intrinsic defenses within the esophageal epithelium and thus results in esophagitis2. Barrett’s esophagus arises in the setting of chronic reflux, and is a premalignant lesion with increased risk of esophageal adenocarcinoma3,4. Despite the clinical importance, the mechanisms of GERD, Barrett’s esophagus and adenocarcinoma have not been well understood.
Animal models are essential for research on etiology, pathology, molecular mechanisms, prevention and treatment of human diseases. Up to date, various animal models of GERD, Barrett’s esophagus and adenocarcinoma have been developed using model animals5,6. Mouse esophagus is lined with stratified squamous epithelium which is histologically similar to that in human esophagus. Although a mouse esophagus is different from human esophagus in terms of keratinization and the absence of submucosal glands, the mouse is still an appealing model animal because of its relatively low cost of maintenance and its potential of sophisticated genetic modifications. Two approaches are commonly used to model GERD, Barrett’s esophagus and adenocarcinoma in mice: reflux surgery and genetic modification. Reflux surgery is the best way to induce reflux and genetic modifications mimics molecular alterations5,7. Reflux surgery can be combined with genetic modifications to further understand disease mechanisms8.
Many surgical procedures have been reported by us and others6,9: (1) gastric reflux: pyloric ligation, pyloric constriction with forestomach ligation, Wendel cardioplasty, and esophagogastric anastomosis; (2) mixed reflux: esophagogastroduodenal anastomosis, esophagoduodenostomy (or esophagojejunostomy); (3) duodenal reflux: esophagogastroduodenal anastomosis plus gastrectomy; (4) reflux of chemical components: bilious reflux, pancreatic reflux, esophageal perfusion; and (5) esophageal transplantation5. Recently a microsurgical mouse model was reported to produce jejunal reflux via an esophagojejunostomy with magnets10. These surgical models have advantages over in vitro cell culture or organotypic culture models. In vitro, esophageal cells cannot tolerate a medium with high acidity or high concentrations of bile acids. Unconjugated bile acids which are commonly used to produce changes in esophageal epithelial cells in vitro are usually not present in the duodenal refluxate in vivo. Thus conclusions drawn from such in vitro studies should be taken with caution.
Surgery on the mouse esophagus remains a technical challenge because of its small size. A low rate of postoperative survival does not allow experiments which require certain sample size to reach statistically sound conclusions. In the past we have successfully developed and characterized surgical models of gastric reflux, mixed reflux, duodenal reflux with mice in long-term experiments9,11,12. We have also provided consultation to several other groups in their mouse surgery. Herein, we describe three surgical procedures in mice in order to help the community to establish these models in their labs.
All the animal experiments have been approved by the Institutional Animal Care and Use Committee.
1. Mouse Preparation
2. Gastric Reflux Model (Figure 1B)
3. Mixed Reflux Model (Figure 1C)
4. Duodenal Reflux Model (Figure 1D)
5. Post-surgical Treatment
Most mice (>95%) can survive the surgery. During the perioperative period, the leading causes of death include overdose of anesthetics, bleeding and unknown reasons.
Four weeks after surgery, >90% mice with gastric reflux or mixed reflux and >80% mice with duodenal reflux can survive. During this period, mice primarily die of esophageal stricture and inability to eat. These mice show signs of severe stress (hunched posture, inactivity, vomiting, sunken eyes, vocalization, etc.) and need to be euthanized. Medical treatment with antibiotics and infusion usually cannot save these mice from dying. Mice usually lose their body weight by a couple of grams after surgery, especially those with duodenal reflux. Nevertheless body weight will recover and increase later on.
Forty weeks after surgery, >80% mice with gastric and mixed reflux and >70% mice with duodenal reflux can survive. During this period, some mice may die from malnutrition or unknown reasons. We have kept mice for 80 weeks after surgery. In general, they are still quite active and healthy9.
At four weeks after surgery, the architecture of esophageal epithelium will be damaged more or less by reflux. Under microscope, the epithelium becomes thicker and the papilla elongates. Hyperproliferation of epithelial cells and infiltration of inflammatory cells (e.g., neutrophils, mast cells and eosinophils) are obvious signs of esophagitis (Figure 2). Cytokines in esophageal epithelium (e.g., IL1β, IL6 and IL8) are elevated particularly in the presence of duodenal reflux11. Under transmission electron microscope, dilated intercellular space reflects the impairment of epithelial barrier function12. Function-wise, trans-epithelial electrical resistance is dramatically decreased, especially in the presence of duodenal reflux.
Figure 1: (A) Anatomy of the upper digestive tract in mice (front view and side view); (B) Gastric reflux (side view); (C) Mixed reflux (front view); (D) Duodenal reflux (front view).
Figure 2: Histology of mouse esophageal epithelium without reflux (A), or with gastric reflux (B), mixed reflux (C), duodenal reflux (D). Scale bar = 50 µm.
Various surgical models have been established to mimic gastric, duodenal and mixed reflux in rodents. These three procedures described here are suitable for long-term experiments with reasonable rates of postoperative survival. A researcher with surgical experience should be able to grasp the technique after a short period of practice.
Bleeding may result from intraperitoneal injection of anesthetics before surgery, laceration of the liver during separation of the connective tissues between the liver and the stomach, and inadvertent damage of the blood vessels. This should be avoided as much as possible. Battery-operated mini-cautery may be used to stop bleeding if appropriate. Excessive stretching should be avoided as much as possible when organs and tissues are manipulated. The tips of the tweezers may be wrapped with cotton, or cotton tips may be used like chopsticks to replace tweezers. The mouse esophagus has two layers or two “tubes”, outer reddish “muscle tube” and inner whitish “epithelium tube”. When an incision is made on the esophagus, both layers need to be cut open. Sutures should be evenly spaced with accurate mucosal to mucosal opposition. Leakage may result from a wide distance between the sutures, whereas stricture may develop due to a narrow distance between the sutures. However, stricture may block the passage of food and cause postoperative death, whereas slight leakage may be fixed by local adhesion and fibrotic reaction and thus is usually not life-threatening.
There are several critically general principles to follow within the protocol. The duration of surgery should be shortened if possible (about 20-30 min in the lab). Fasting before and after surgery is unnecessary. In fact, prolonged fasting (>1 day) may be harmful. Surgery is best performed by one surgeon. An assistant may help anesthetize the mice and supply surgical materials. A microscope is not necessary unless the surgeon has been properly trained in microsurgery. Otherwise the duration of surgery tends to be longer than necessary and thus postoperative survival may be potentially worse.
These procedures have been used in previous publications and have generated satisfactory results. Using wild-type and genetically modified mice, we demonstrated that NFκB and Nrf2 pathways regulated the barrier function of esophageal epithelium during gastroesophageal reflux11,12. However, we were not able to produce full-blown Barrett’s esophagus or adenocarcinoma in these mice, instead, scattered mucinous cells and squamous cell carcinoma were induced9. Histological differences between mouse esophagus and human esophagus may be the inherent limitations of these surgical models in research on Barrett’s esophagus and esophageal adenocarcinoma.
We believe these surgical models are useful for studies on GERD. Functions of genes and molecular pathways can be elucidated to understand the molecular mechanisms of GERD and develop mechanism-based therapy. When used for producing Barrett’s esophagus and esophageal adenocarcinoma, genetic modifications will be needed in combination with reflux surgery.
The authors have nothing to disclose.
We are supported by research grants from the National Natural Science Foundation of China (NO. 81400590), National Institutes of Health (U54 CA156735) and Takeda Pharmaceutical Company Ltd. (MA-NC-D-156).
Instruments | Source | ||
Dumont #1 Forceps Dumostar Tip | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Micro Clip Applying Forceps 5.5" | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Bonn Scissors 3.5" Straight 15mm Sharp/Sharp Tungsten Carbide Blades | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Operating Scissors 5.5" Straight Sharp/Sharp SureCut | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
4-0 Silk Black Braid 100 Yard Spool | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Surgeon's Needle 1/2 Circle Cutting Edge Size 12 (25 mm Chord Length) Pack 12 | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Halsey Needle Holder 5" Smooth | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Micro Needle Holder 5.125" Curved Lock .6mm | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Reflex 9mm Wound Clip Applier | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
Reflex 9mm Wound Clips Box Of 100 | Roboz Surgical Instrument Co. (Gaithersburg, MD) | ||
PRONOVA Poly (hexafluoropropylene-VDF) Suture 8-0 | Ethicon US, LLC | ||
Reagents | Source | ||
Ringer's solution | Henry Schein, Inc. | ||
ketamine | Henry Schein, Inc. | ||
xylazine | Henry Schein, Inc. |