胃食管反流与小鼠手术模型

摘要

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.

摘要

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.

引言

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.

研究方案

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

1. 过使用小鼠约20克体重或以上〜6周龄进行手术。
2. 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。
3. 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序。
4. 确认基于无角膜反射和肢体回缩适当的麻醉，当脚垫被挤压。应用润滑油眼药膏的眼睛，以防止干燥。
5. 皮肤消毒用聚维酮碘溶液。

2.胃反流模型（图1B）

1. 使上的〜2厘米从剑突指向肛门起始中线切口。
2. 通过中线打开腹腔。删除剑突，提升曝光剪刀。
3. 分开和割肝和胃之间的结缔组织。结扎和切断脾和眼底的血管束，以完全自由了眼底。
4. 转动眼底稍微向左和胃食管接头的左侧露出在该领域。使上沿采用一对锋利的剪刀，露出上皮食管远端肌肉5mm的纵形切口。
5. 切割沿相同的方向（切口1， 图1B）打开上皮。使三切口上forestomach（切口2,3和4， 图1B），以切断大部分forestomach与尖锐操作剪刀。如果胃不空，小心地取出胃内容物。
6. 将8-0普理灵苏图重（带锥形尖的针）通过一个点（在食管）和点A'（在forestomach）准确粘膜粘膜反对。同样的地方缝合线通过点B和点B'，以及其他对点，分别为。均匀空间这些缝线3-4毫米彼此。
7. 用生理盐水冲洗腹腔，清理血液和胃内容物。关闭腹壁丝线缝合并用金属夹皮。

3.混合反流模型（图1C）

1. 按照步骤2.1至2.4，露出胃食管交界处。
2. 轻轻分开食道的背侧从食道后面的血管。通过食管和血管之间的小棉签。
   注意:棉可能从尖被部分地去除，以减少其尺寸。此棉尖端有两个目的，举起食道和保护血管。
3. 做两个5毫米纵向INC过程中反冲每个上胃食管接头和十二指肠相邻尖锐操作剪刀幽门近端。对十二指肠的切口，避免血管和放置在反肠系膜边界。
4. 吻合准确粘膜切口粘膜间断8-0普理灵缝线反对。一般放置3-4缝线上背侧和2-3缝线上的前侧。
5. 取出棉签。冲洗腹腔用生理盐水并关闭腹壁和皮肤。

4.十二指肠反流模型（图1D）

1. 按照步骤3.1到3.4，以生成混合回流。
2. 小心地抬起肚子，露出它的背面。肝脏的叶可被捕获的肝和胃的背面之间结缔组织。小心切割结缔组织，妥善保护肝脏，并露出血管的食道的背侧。
3. 结扎和切割血管。结扎并切断食道在胃食管接头。
4. 结扎十二指肠的幽门。结扎并切断肠系膜。删除整个胃。
5. 用生理盐水冲洗腹腔。关闭腹壁和皮肤。

5.手术后治疗

1. 手术后保持体温有一个加热垫。只有恢复胸骨斜卧后，将小鼠背部与其他动物。
2. 给予抗生素和止痛药，以预防感染，减轻疼痛。注入拜有利（10毫克/ kg，腹腔，连用 ，3天），以防止感染，而丁丙诺啡盐酸盐作为止痛药（0.05毫克/公斤，SC，出价。两天）。手术后禁食是没有必要的。
   液体或软食，可给予。
3. 评估日常basis.If的小鼠表现出下列迹象的一般健康状况（戏剧性的体重减轻> 15％，缺乏应用的etite，发声，从口，鼻或眼睛，弯腰驼背的姿态，不活动，不正常的疏导活动）排出，安​​乐死小鼠。

结果

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?...

讨论

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?...

材料

Name	Company	Catalog Number	Comments
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 15 mm 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 .6 mm	Roboz Surgical Instrument Co. (Gaithersburg, MD)
Reflex 9 mm Wound Clip Applier	Roboz Surgical Instrument Co. (Gaithersburg, MD)
Reflex 9 mm Wound Clips Box Of 100	Roboz Surgical Instrument Co. (Gaithersburg, MD)
PRONOVA Poly (hexafluoropropylene-VDF) Suture 8-0	Ethicon US, LLC
Ringer's solution	Henry Schein, Inc.
ketamine	Henry Schein, Inc.
xylazine	Henry Schein, Inc.