鼠回结肠肠管切除原发性吻合

摘要

Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease.

摘要

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn’s disease and colon cancer being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease.

引言

Ileocolic resection (ICR) is a common procedure performed in both emergent and elective situations for a variety of illnesses. Crohn’s disease and colon cancers are the two most common indications for ICR. In both illnesses, recurrence in the bowel at the site of surgery represents a major clinical problem. Local recurrence rates for colon cancer remain an issue even with the most aggressive resections¹. Following ICR in Crohn’s disease, the disease most frequently (in up to 80%) recurs in the neo-terminal ileum at 1 year after surgery². Given the impact of these two illnesses and their recurrence after surgery, it is important to understand local intestinal factors after ICR, which may have intrinsic influences on the natural history of these diseases. Further, it is also important to consider anastomotic healing after ICR. In the early post-operative period, anastomotic leaks can have devastating consequences for patients resulting in repeat surgeries, stoma creations, significant morbidity, and even mortality³. Despite the importance of this topic, our current understanding of anastomotic healing remains in its infancy as a subject of research. Animal models of ICR, in particular mice, are an excellent platform for studying the intestinal and anastomotic healing following surgery.

A mouse model of ICR was initially developed by Helmrath et al. to be used as a model of short gut syndrome⁴. The authors experimented with various diet regiments and suture sizes to optimize animal survival following ICR. They concluded that feeding with liquid diet in the perioperative period and using 9-0 monofilament sutures resulted in an optimal post-operative survival of 88%. Since this initial publication, ICR in mice removing 50% of the small bowel has been used in several studies to explore the dynamics of massive small bowel resection and the adaptive growth response in attempt to develop new therapies for short gut syndrome^5,6.

The first application of the ICR mouse model to Crohn’s disease used the IL-10^-/- mouse model, which spontaneously develops colitis⁷. The authors found that after ICR these animals developed inflammation in the neo-terminal ileum similar to that seen in post-operative Crohn’s disease patients, and that this inflammation was dependent on the presence of bacteria⁷. More recently, this model was used to explore bacterial changes induced by ICR. In Crohn’s disease there is an associated dysbiosis with relative decreases in bacteria known to have anti-inflammatory properties and increases in invasive species of bacteria^8,9. The association holds true in cases of post-operative recurrence¹⁰. Two studies sought to identify microbial changes resulting from ICR. The first used IL-10 null mice, and performed denaturing gel electrophoresis to compare bacterial similarity between the small bowel and colon after ICR¹¹. This study demonstrated that bacterial populations became similar in the small intestine and colon following ICR. A subsequent study used wild type mice and 16s pyrosequencing for phylogenetic classification of bacterial species post-operatively. This study demonstrated a marked shift in bacterial species resulting from surgery alone with Clostridium species becoming dominant as well as an increase in ϒ-proteobacteria. The results also confirmed the findings of the previous study with similar populations found in the small bowel and colon after ICR¹².

ICR is a common procedure for patients with colon cancer involving the cecum and ascending colon, and it is becoming increasingly recognized that the host-response to surgery likely contributes to both local and distant tumor recurrence¹³. Despite this observation, models of ICR have not been utilized for the study of colorectal cancer and post-operative recurrence. Understanding both the systemic and local immunologic changes resulting from ICR will be important in investigating future therapies. Potential pathways involved in cancer recurrence post ICR include up regulation of growth factors, which may rescue cells from apoptosis and stimulate proliferation, mechanical tumor disruption with cell shedding, and loss of immune surveillance through post-operative immunosuppression^13,14.

Mouse models of ICR have the potential to be a powerful tool for the investigation of short bowel syndrome, Crohn’s disease, and colon cancer. They may also provide lessons on how to prevent early post-operative anastomotic complications by further defining the cellular and biochemical pathways involved in healing the newly constructed anastomosis. A major barrier in utilizing the murine ICR model is the technical difficulty. The intestinal anastomosis requires the use of 8-0 or 9-0 suture, an operating microscope, and training in microsurgical techniques. The goal of this article is to provide clear instructions on how to perform ICR in mice with the goal of utilizing this procedure in models of disease.

研究方案

动物使用协议批准了在阿尔伯塔大学的健康科学动物管理和使用委员会。

1.准备仪器，动物和手术的设置

1. 动物转移到之前的程序所有的固体食物24小时的一个新的，干净的笼子里缺席。他们可能有自由饮水，并流质饮食即兴直到该过程的时间。
2. 高压灭菌所需的程序的所有仪器。清洁操作表面和麻醉剂的鼻锥，用70％的乙醇。
3. 设置的方式，这是舒适，手术医生用手术显微镜，麻醉机和用品的操作表面。外科医生的肘部应允许舒适地休息，在手术台上，用双手和双臂受通畅设备。器械，缝线，棉签，和10ml的注射器应该被放置在一个位置，它允许在操作过程中便于使用。
4. 建立开销加热灯，以提供温暖的程序和光的操作表面中的动物。
5. 填在50ml的锥形管中以0.9％的盐水，和1.5ml试管用凡士林和场所的操作表面附近。
   注:所有的消毒仪器和手术用品。因为肠道被横切，该过程本身是不育的。它被认为是干净的污染。采取措施，避免引进感染的外源性来源。

2.回结肠切除吻合术

1. 通过施用4％异氟烷以2升/分钟通过鼻锥从异氟烷蒸发器的氧流量诱导麻醉，直到动物呼吸速率减慢到大约30-40次/分。施加适当的压力，以鼠标的后足，以确保有之前启动过程没有疼痛反应。在这一点上，调低异氟烷至2％，氧流量0.5升/分。间歇在此过程中检查的疼痛反应，并相应地调整异氟醚的流量。
2. 适用凡士林眼睛，以防止干燥手术过程中，并固定在鼠标的四肢用透明胶带固定仰卧位。
3. 请用povodine /碘溶液的腹部，并转变成新的无菌手套。
4. 让1.5厘米皮肤切口用锋利点解剖剪刀，以暴露筋膜和腹膜腹部的上部中线。通过白线打开以类似的方式筋膜/腹膜层腹膜内容露出。
5. 而相比之下，人类，小鼠盲肠通常位于腹部的左上象限。一旦确定，轻轻抓住钳盲肠和通过切口提供它。用浸湿的棉签扇出约3cm回肠末端与盲肠在一个无菌纱布搭在腹部表面（ 图1A）延伸。确保该过程的全部过程中暴露的肠保持湿润以0.9％的盐水。
6. 确定回盲动脉分支沿结肠（ 图1A）的肠系膜上动脉使用手术显微镜。剖析了相邻的回盲动脉的缺血性组织，包围并结扎用5-0丝线扎动脉。接下来，找到区域的血液供应，回肠末端，然后选择一个横切点1.5-2厘米近端回盲部。结扎分支为上述这部分回肠。划分微型解剖剪刀动脉。
7. 分回肠和结肠确保有足够的血液供应，切断端部（ 图1B）的缺血部分。常常是有帮助通过它除以一个30度角，以增加腔的直径，从而它更紧密地结肠匹配到匙形回肠。一旦肠道回盲部已被删除，对齐回肠和结肠的纱布切断端部，从而确保每一个的肠系膜的边界对齐。
8. 通过近似回肠的横切端使用结肠横切端构造吻合间断8-0聚丙烯缝合线（ 图1C）。所述第一线圈被放置在肠系膜边​​界，随后缝线置于每0.5毫米直到回结肠吻合术是水密的。当通过回肠和结肠缝合针，保证切割边缘不卷，和针叮咬为0.5毫米肠道的切割边缘。一个典型的吻合将需要14至16间断缝合。通过轧制棉签近端至远端在回肠通过吻合迫使内容完成时测试该吻合的完整性和通畅。小肠内容应自由地传递到不吻合口漏的结肠。
9. 从10毫升注射器冲洗暴露的肠用3-4毫升0.9％盐水中的洗去粪便从肠的表面上，并提供肠放回腹腔。使用2毫升的0.9％盐水冲洗腹腔，然后通过施加轻微的压力于腹壁横向排水该流体。
10. 关闭切口用3-0丝缝合线的运行，并终止异氟醚的流动。施用0.1毫克/千克的长效鸦片-buprenorphine-皮下手术后疼痛控制。
11. 观察下加热灯的动物，直到他们的移动，然后将它们转移到一个不断升温的笼子。

3，术后护理和监测

1. 监测动物在不断升温笼窘迫的迹象了一天的剩余部分。动物转回到了动物护理设施的新的无菌笼子访问流质饮食和水的即兴表演。动物可以被容纳在3-4组。
2. 在手术后的动物进行检查，第二天早上，确保动物不会出现遇险。只喂流质饮食。如果出现不舒服（ 例如，弯腰驼背的姿势或活动极少）管理皮下丁丙诺啡的附加 ​​剂量。检查动物再次在下午手术后第1天。
3. 在手术后第2天上午的动物应该出现全面复苏。食品消费和stooling的证据是复苏的积极迹象。现在，恢复了坚实的食物饮食的动物。
   注:窘迫的症状包括驼背的姿势，疏导差，最小的活动。如果窘迫的迹象很突出的动物应该被安乐死。
4. 通过诱导用4％异氟烷深度麻醉，以2升/分钟的O ₂流量，直至动物安乐死的动物是不响应足压。进行颈椎脱位，并遵守正确安乐死的迹象。
5. 指引而有所不同，所以请参阅有关指示机构的建议对于和安乐死的小鼠的适当方法。

结果

死亡率和体重变化术后。
在129S1野生型小鼠以下ICR死亡率一般〜5％。道德的最常见原因是肠梗阻的吻合。死亡的其他原因包括吻合口瘘和内疝，导致肠梗阻。

体重减轻可以看出长达14天手术后，但一般不显著。小鼠倾向于通过手术后第28天（ 图2）完全恢复手术前的重量。

技术的可转让性
新的研究生（BM）被教导要执?...

讨论

鼠的ICR是可以用来研究手术中肠疾病的效果的强大的模型。本文介绍用95％的成功率和失败的蓬勃发展为以稳定的权重至28天手术后没有反映出来的问题进行ICR小鼠的方法。成功ICR最显著的挑战包括避免肠梗阻的吻合及吻合口漏。

技术要素的手术，以防止阻塞的目的是在吻合最大化内腔直径。回肠在横断点Spatulation通过将肠在如在图1A中所示的30度角增大管腔直径?...

材料

Name	Company	Catalog Number	Comments
LD101 liquid rodent diet	testdiet.com
0.9% NaCl	Baxter	FKE1324	Injection quality saline
Operating Microscope	Ziess
Isoflurane Anesthetic Vaporizer	Harvard Apparatus	34-0483
Isoflurane	Abbott	05260-05
Glass plate			For operating surface
Cotton swabs
Micro Castroviejo Needle holder, curved	World Precision Instruments	503377
Castroviejo straight scissors	World Precision Instruments	555530S
Dissecting Scissors	World Precision Instruments	15922
Dressing Forceps x 2	World Precision Instruments	500363
5-0 silk pre-cut sutures	Ethicon	A182H	For vessel ligation
8-0 Prolene on BV130-5 needle	Ethicon	8732H	For anastomosis
3-0 Silk on FS-2 needls	Ethicon	8665G	For abdominal wall closure
Petroleum Jelly	Vaseline
10 ml syringe	BD biosciences
Povidone-iodine 7.5% surgical Scrub	betadine.com
Heat lamps
buprenorphine 0.3 mg/ml	Reckitt Benckiser Healthcare Ltd.	PL36699/0006