在鼠标空肠和结肠段肠系膜传入神经活性的体外记录
Summary
Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.
Abstract
传入神经不仅传达信息有关正常生理，而且信号干扰稳态和从向中枢神经系统周围的不同器官系统的病理生理过程。这样，增加的活性或肠系膜传入神经的“敏化”已被分配在内脏高敏感性和腹痛综合征的病理生理学中起重要作用。
肠系膜传入神经活性可以在体外 ，其安装在一个特制的器官浴中，并从该内脏神经是隔离的，使研究人员能够直接评估邻近胃肠段神经活性的分离的肠段来测量。活性可在标准条件基线段的膨胀或增加intraluminally或serosally递送药理化合物以下期间被记录。这种技术允许研究者容易学习的药物靶向于对照样品外周神经系统的影响;此外，它提供了对疾病的过程中是如何的神经元活动改变的关键信息。但是，应该注意的是，测量传入的神经元放电活动只构成了复杂的神经元一个中继站的信号级联反应，研究人员应该牢记没有其他级别（ 例如，背根神经节，脊髓或中枢神经系统忽视的神经元活动）为了充分阐明在健康和疾病的复杂的神经生理学。
常用的应用包括神经元活动的响应于脂多糖给药研究中，和传入神经活性的肠易激综合症的动物模型的研究。在一个更平的方法中，分离的小鼠肠道段可暴露于来自IBS患者结肠上清。此外，修改这种技术的最近已经证明是适用于人结肠标本。
Introduction
感官信令和疼痛知觉是一个复杂的过程，从传入神经，脊髓神经元，升序和降序易化和抑制途径和几种不同的脑区域之间的错综复杂的相互作用的结果。正因为如此，在一个或多个这些水平的变化可能导致改变的感觉的信号和在疾病状态内脏疼痛。研究的感官信令多种技术所有这些不同的方面已经开发了从单细胞实验（ 例如，在神经元的钙成像）到整个动物模型（ 例如，行为反应，如内脏运动响应）。本文描述的技术可以让研究人员专门从啮齿动物的小肠或结肠的一隔离段评估体外传入神经活动。总之，分离的胃肠道段（通常空肠或结肠）安装在用生理ķ灌注一个专用记录室篮板的解决方案。内脏神经解剖自由和连接到允许传入的神经元活动的登记内脏或盆腔传入神经的电极。神经活性可以基部或响应于，或者通过管腔内灌洗液（粘膜）提高腔内的压力和/或药理学化合物可直接施加到记录室（serosally），以评估其对传入放电1-6效应被记录。值得注意的是，内脏神经还含有传出纤维和除了感觉传入viscerofugal传入。一种体外内脏神经记录的一个主要优点是，研究人员可以量化神经活动而不脱离中枢神经系统调制或输入，使一个以研究局部施加化合物对神经活动的直接作用的事实。此外，重要的参数的监控，如使用体内方法是必要的（见下文），为正Ø不再相关。 体外内脏记录终于要少得多耗时比其在体内的对应。
响应于其他刺激，如粘膜划线，使用von Frey毛探测或段的拉伸传入神经元活动，可以在修改的实验装置，其中肠组织被牵制和纵向打开（这是相对于被研究我们的设置使用一个完整的片段），如在先前的问题7,8进行了说明。此外，仅在最近，这种技术被描述来研究通过钙成像结肠壁本身结肠传入神经激活，再次使用牵制，纵向打开段9。
这种体内技术的另一种版本包括了测量传入的进入脊髓邻近神经元激活的。总之，镇静动物置于俯卧位，例如xposing腰骶脊髓通过椎板切除术的手段，构建感兴趣的项目的该传入神经石蜡填充孔用切口的皮肤和悬垂在铂双极电极10,11背侧支根。这种技术还使研究人员能够从薄髓Aδ纤维表征基于其传导速度纤维和无髓鞘区分C纤维。此外，背侧支根只包含感觉传入纤维，而相比之下，前面提到的混合传入和传出内脏神经。
从孤立的肠道段录音在体外传入神经放电，也可以使用人体标本进行，如独立出版首次在男子手稿记录在人类结肠切除术传入纤维的活动标本12,13两个研究小组。该技术的实施可能会导致更容易translati对小鼠的数据，以人性化的状态，并且可以让研究人员能够轻松识别靶向药物致敏的感觉神经。表征传入神经活动，以及为针对过高的传入神经活动的新治疗剂的发现的临床重要性，一直精心许多专家在现场讨论14-19。
上述体外技术补充了在传入神经活动的体内测量更多的俗称。期间在体内神经元活性的测量，神经活性可以直接在其间感兴趣的段被识别，并随后插管的镇静动物测定，和液体石蜡填充井使用啮齿类20的腹壁和皮肤构成。然后感兴趣的传入神经被识别，切片并放置于双极铂电极，使神经元活动measuremen吨。这种技术允许研究人员来调节生活虽然镇静动物传入纤维的活动;因此，可以研究神经元活动响应干扰诸如鲁米腹胀或化合物的静脉内给药。
转化研究现在主要集中在人衍生的上清液中的应用（ 例如 ，从结肠活检，栽培外周血单核细胞等 ）上空肠和/或结肠鼠标传入21,22。研究人员可以直接申请上清要么进机关浴缸或成灌注到段肠管腔内的解决方案，因此浆膜与粘膜应用的不同影响可以传入神经放电进行研究。因此，它表现出从患者肠易激综合症结肠黏膜活检supernatans可导致小鼠结肠传入，豚鼠粘膜神经元和小鼠背根超敏反应神经节神经元21,23,24。
最后，记录神经元的活动并不限于肠系膜和/或骨盆神经支配胃肠道。他人已经证明，神经录音可以在传入供给膝关节25来执行，而另一些特征膀胱传入神经活性以及26-28，并表明从膀胱骨盆传入以及胃肠道汇合，从而可能导致神经元串扰29。
Protocol
Representative Results
Discussion
本文的协议描述一个可重复的实验技术13759我们组和其他3,4,7,8,12,20,21,31研究啮齿动物的肠系膜传入神经活动。在协议中的关键步骤包括组织的快速分离，神经链的抽吸进入抽吸电极和通过吸周围脂肪组织进入毛细管从器官浴中的玻璃毛细管的适当“密封”。玻璃毛细管的孔径应当精确确定：太小的孔将神经链的抽吸复杂化到电极，而太宽孔径会阻碍与脂肪组织的毛细血管的“密封”，从?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
SN performed the experiments described above, performed the data analysis and drafted the manuscript. AD and JDM implemented the technique at our research facilities and aided in the data analysis. HC aided in performing the experiments. WJ, CK and DG assisted in implementing the afferent measurement technique in our lab, the data analysis and interpretation of the results. SF, JDM and BDW designed the study. All authors critically read and approved the final manuscript. SN is an aspirant of the Fund for Scientific Research (FWO), Flanders (11G7415N). This work was supported financially by the FWO (G028615N and G034113N).
Materials
| sodium chloride (NaCl) | VWR Chemicals | 27,810,295 | compound Krebs solution |
| potassium chloride (KCl) | Acros organics | 196770010 | compound Krebs solution |
| sodium dihydrogen phosphate (NaH2PO4) | VWR Chemicals | 1,063,461,000 | compound Krebs solution |
| sodium bicarbonate (NaHCO3) | Merck | 1,063,291,000 | compound Krebs solution |
| magnesium sulfate (MgSO4) | Merck | 1,058,861,000 | compound Krebs solution |
| calcium chloride (CaCl2) | Merck | 23,811,000 | compound Krebs solution |
| D-glucose | VWR Chemicals | 1011175P | compound Krebs solution |
| Distilled water | compound Krebs solution | ||
| PVC tubing | Scientific Laboratory Supplies | The intestinal segment should be mounted over PVC tubing | |
| Silicone tubing | Scientific Laboratory Supplies | The rest of the tubing, ideally silicone-based – more easily dislodging of debris in the tubing | |
| Silk thread | Pearsall Limited | 10B15S220 | Attachment of the segment over the PVC tubing |
| Syringe driver | Harvard Apparatus | 55-2222 | Intraluminal infusion of Krebs |
| Binocular – including 10x magnification in oculair | Zeiss STEMI 2000 | Optimal visualization for the dissection of the afferent nerve | |
| Homeothermic Blanket Control Unit | Harvard Apparatus | 507214 | Heating of the organ chamber |
| Custom made organ bath with Sylgard covered bottom | |||
| Spike2 software | Recording and analysis of the data | ||
| Insect pins, 500 pieces, stainless steel, diameter 0.2 mm | Austerlitz insect pins minutiens | Dissection of the afferent nerve | |
| Tweezer Dumont #5 inox 11cm | World Precision Instrument | 500341 | Dissection of the afferent nerve |
| Scissors, spring, 14 cm | World Precision Instrument | 15905 | Dissection of the afferent nerve |
| DB digitimer | NL 108T2/10 | pressure transducer | |
| Micromanipulator | Narishige | M-3333 | 3D manipulation of the suction electrode |
| Micromanipulator | X-4 rotating block | 3D manipulation of the suction electrode | |
| Micromanipulator | GJ-8 magnetic stand | 3D manipulation of the suction electrode | |
| LightSource | Euromex Microscopes Holland EK-1 | Optimal visualization for the dissection of the afferent nerve | |
| CED 1401 Recording Apparatus | Recording of afferent nerve activity | ||
| Humbug 50/60Hz Noise Eliminator | Quest Scientific Instruments | Elimination of background noise | |
| Infusion Pump | Gibson Minipuls 2 | Infusion of the organ chamber in which the segment is mounted | |
| Microelectrode Holder Half Cells 1.5 mm | World Precision Instrument | MEH2SW | Suction electrode for isolation of the afferent fiber |
| Borosilicate Glass Capillaries, 300 pc; 1.5/0.84 OD/ID | World Precision Instrument | 1B150-4 | Capillary for the isolation of the afferent nerve |
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