胃食管反流与小鼠手术模型

Summary

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.

Abstract

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.

Introduction

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 adenocarcinoma^3,4. 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 animals^5,6. 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 alterations^5,7. Reflux surgery can be combined with genetic modifications to further understand disease mechanisms⁸.

Many surgical procedures have been reported by us and others^6,9: (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 experiments^9,11,12. 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.

Protocol

所有的动物实验已批准的机构动物护理和使用委员会。 1.鼠标准备过使用小鼠约20克体重或以上〜6周龄进行手术。 手术前，给小鼠实验室饲料自由采食和饮水，并在12:12小时的明暗周期维护。 剃了头发剪手术区。通过腹膜内注射80毫克/千克氯胺酮和12毫克/千克甲苯噻嗪麻醉的小鼠。该剂量使小鼠进入睡眠在几分钟后，并提供足够的麻醉以下手术程…

Representative Results

大多数小鼠（> 95％）可以存活手术。在围手术期，导致死亡的原因包括过量麻醉药，出血及原因不明。 四周手术后，> 90％的小鼠胃返流或混合反流和> 80％的小鼠十二指肠反流可以生存。在此期间，主要小鼠死于食管狭窄和无法吃。这些小鼠表现出严重的压力（驼背的姿势，不活动，呕吐，眼睛凹陷，发声等 ）的迹象，需要进行安乐死。用抗生素及输液药物治?…

Discussion

各种手术模型已经建立了模拟胃，十二指肠和混合回流的啮齿动物。这里介绍这三种方法都适用于术后生存的合理的价格长期的实验。与手术经验的一位研究人员应该能够练习后短期内掌握的技术。

出血可能源于腹膜内注射麻醉剂的手术，肝和胃，和血管无意损害之间的结缔组织的分离期间肝脏裂伤之前。应该避免这种情况尽可能。电池操作的微型烧灼可被用于停止，如果?…

Materials

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.