视频成像和时空地图来分析胃肠动力小鼠
Summary
This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.
Abstract
肠神经系统（ENS）在调节胃肠（GI）运动中起重要作用，并且可以独立于中央神经系统的作用。在ENS函数变化的胃肠道症状和疾病的主要原因，并可能有助于在神经精神障碍包括自闭症报道胃肠道症状。它是公认的离体结肠段产生被称为结肠移植电机配合（CMMCs）自发的节律性收缩。一个过程来分析前说明小鼠结肠的体内筹备CMMCs的肠道神经调节。结肠从动物解剖并冲洗以在器官浴中插管之前除去粪便内容。数据经由定位在器官浴上方的摄像机获取的，并通过一个内部软件包转换成高分辨率的时空图。使用这种技术，基线收缩模式和结肠S于ENS功能的药理作用egments可以在3-4小时进行比较。此外，CMMCs的传播长度和速度可被记录，以及在肠道中直径和收缩频率的变化。这种技术是用于表征在转基因小鼠模型（以及在其它物种，包括鼠和豚鼠）胃肠运动模式是有用的。以这种方式，在CMMCs药理学诱导的变化被记录在野生型小鼠和孤独症的人类neuroligin-3 R451C小鼠模型。此外，这种技术可应用于胃肠道的其他区域，包括十二指肠，空肠和回肠，并在小鼠中不同发育年龄。
Introduction
肠神经系统（ENS）是胃肠道的固有的神经元网络，并调制各种功能，诸如肠内容消化，营养吸收和分泌和流体的重吸收。在ENS的神经元位于肌间和粘膜下神经丛。肌间神经丛在调节肠胃蠕动1，而黏膜下神经丛的一大作用是主要参与分泌2,3的控制。肌间神经丛坐落于胃肠壁的纵向和环形肌层之间。肠壁的平滑肌层的收缩活性通过混合和推进沿着肠管3的长度肠内容便于胃肠道的主要功能。虽然非本征神经供应到胃肠道从中枢神经系统有助于体内胃肠功能中，ENS是能够独立调节胃肠功能。这种独特的特性使肠神经回路的功能调查，他们对肠胃蠕动体外贡献。
结肠移行络合物（CMMCs）是在缺乏粪粒4-9的离体小鼠结肠中观察到的主要电机图案自发的，神经性的事件。 CMMCs被定义为沿着水平距离，该距离至少结肠的总长度的一半传播（即，从盲肠到直肠）10节律性收缩。 CMMCs并且推进粪粒的收缩型态之间的关系尚未清楚地确定，但是一些药理差异已经报道11。尽管如此，ENS的中枢神经系统独立工作的能力和神经介导的运动模式在IS中存在olated结肠提供了一个理想的检测系统来检查的运动障碍，从根本ENS功能障碍所致。胃肠运动模式的自发性允许响应于药理刺激功能的改变进行评估。
使用视频成像和时空映射首次制定定量检查豚鼠12小肠道蠕动。在这里， 离体技术进行说明，使小鼠结肠动力模式的研究利用视频成像和这些录音的分析，构建高分辨率（〜100微米，33毫秒）结肠直径地图作为位置沿结肠功能和时间（时空地图）的。利用内部边缘检测软件（Analyse2;应要求提供），由全长结肠段实时承包数据进行处理，生成每个实验时空地图。在此步骤中，视频格式（AVI）文件是最优等授权的，并转换成使用Analyse2时空映射。时空映射（图2）描述了收缩随时间和使多个参数，包括传播速度，大小，长度和持续时间的测量。肠直径也被记录在整个实验作为组织段的整体收缩的量度的持续时间。这种方法可以适用于识别在收缩配合这可能表明改变的肠神经连接的起始点的差别。
旨在评估在豚鼠颗粒推进类似的视频成像协议已经报道13但是在这里我们概述了自发结肠运动的量化的视频成像方法的应用程序（即在没有颗粒）。我们还提供了详细的资料，以协助清扫和准备胃肠组织上的视频成像的方法。本协议提供了用于分析在疾病的动物模型包括遗传小鼠模型肠胃功能的肠溶神经控制的可访问和容易复制工具的研究人员。
视频成像技术使得结肠运动的响应于各种药理学药剂的分析。药物可通过肠腔或器官浴外部结肠制剂给药。鼠标胃肠道的不同区域表现出特定运动模式，如在结肠小肠分割和CMMCs。
此技术已经被用来识别在小肠功能应变的差异;与5-HT 3和5-HT 4拮抗剂差动灵敏度在Balb / c和C57 / BL6小鼠的空肠，观察由于在两种菌株6表示的TPH2基因的多态性。动力上5-HT的抑制的作用仍然CONtroversial，作为冲突的数据已报道内源性5-HT对结肠的蠕动和CMMCs 14,15的重要性。改建动力前和出生后发育7中，以及在疾病10的动物模型肠胃蠕动基因突变的影响，也可以通过利用视频成像检查。在这里，我们说明了结肠运动的自闭症的NL3 R451C小鼠模型，它体现在NLGN3基因编码突触粘附蛋白人类neuroligin-3 16的错义突变研究使用的方法。这种突变是首次确诊患有自闭症谱系障碍（ASD）17，这是强烈胃肠道功能障碍相关的18-22患者识别。我们调查了NL3 R451C突触突变是否会影响使用视频成像技术在ENS神经输出。我们在基线和响应于血清素能5H呈现数据表征CMMCsŧ3/4受体拮抗剂托烷司琼 自闭症的NL3 R451C小鼠模型。
Protocol
Representative Results
Discussion
使用此视频成像技术，CMMC频率测量为结肠运动的野生型的指示和NL3 R451C小鼠，自闭症谱系障碍17的小鼠模型。我们的结果表明在CMMCs中相比，在5HT 3/4受体拮抗剂托烷司琼表明NL3 R451C小鼠表现出增加的敏感性托烷司琼的存在野生型小鼠突变NL3 R451C小鼠数量的减少。因此，我们建议，人类neuroligin -3- R451C突变改变血清素通路，可能通过调节在肠神经元，粘膜或?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
JCB和ELH-Y分别由国防部CDMRP孤独症研究计划的美国能源部（AR11034）的支持。 NHMRC（1047674）到墨尔本的信任ELH-Y.The月斯图尔特助学金，资助大学奖学金MS。我们感谢阿里·塔希尔，法蒂玛Ramalhosa和格拉西亚塞格的技术贡献。
Materials
| Reagents | |||
| NaCl (MW: 58.44) | Sigma-Aldrich | S7653-250G | |
| KCl (MW: 74.55) | Sigma-Aldrich | P9333-500G | |
| NaH2PO4.2H2O (MW: 156.01) | Chem Supply | 471-500G | |
| MgSO4.7H20 (MW: 246.48) | Chem Supply | MA048 | |
| CaCl2.2H2O (MW: 147.02) | Chem Supply | CA033 | |
| D-Glucose anhydrous (MW: 180.16) | Chem Supply | GA018-500G | |
| NaHCO3 (MW: 84.01) | Chem Supply | GA018-500G | |
| Name | Company | Catalog Number | Comments |
| Materials | |||
| Two chambered organ bath Dimentions: 14 cm x 8 cm x 3 cm |
Custom Made | Contact Laboratory Directly | |
| 732 MULTI -PURPOSE SEALANT CLEAR | Dow Corning Australia Pty Ltd | 1890573 | |
| SYLGARD 184 SILICONE ELASTOMER KIT | Dow Corning Australia Pty Ltd | 1064291 | |
| STOPCOCK 3 WAY FEM-ML L/LOCK S | Terumo Medical Corporation | 0912-2006 | |
| SYRINGES with Luer Lock Tips 50mL, 20 mL, 10 mL | Terumo Medical Corporation | N/A | |
| 1.57 mm (ID) x 3.16 mm (OD) – Silastic Tubing | Masterflex | 508-008 | |
| 1.02 mm (ID) x 2.16 mm (OD) – Silastic Tubing | Masterflex | 508-005 | |
| 1.50 mm (ID) x 2.50 mm (OD) – Silastic Tubing | Masterflex | 508-007 | |
| 1.60 mm (ID) – Platinum cured silicone tubing | Masterflex | 96410 – 14 | |
| 4.40 mm (ID) – Platinum cured silicone tubing | Masterflex | 96410 – 15 | |
| 3.10 mm (ID) – Platinum cured silicone tubing | Masterflex | 96410 -16 | |
| Graduated Laboratory Glass Bottles – 500 ml | Thermofisher Scientific | 100-400 | |
| CHEMICAL RUBBER STOPPER 57 x 65mm | |||
| CHEMICAL RUBBER STOPPER 29 x 32mm | |||
| Water heater (thermo regulator) | Ratek | TH7000 | |
| Logitech Webcam | Logitech | ||
| Name | Company | Catalog Number | Comments |
| Software | |||
| Virtual Dub – 1.9 11 | virtualdub.org | ||
| MATLAB R2012a | Graph Pad | ||
| Logitech Webcam Software | Logitech |
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