Name: in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats
Uploaded: 2017-09-27T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

该议定书得到了中国国家自然科学基金 (#31171066、#81270464) 和中德科学中心 (GZ919) 的研究资助。

这里报告的实验协议已获得上海交通大学动物伦理委员会 (SYXK2013-0050) 的批准. #160; 完整的神经节和神经直肠的解剖躯干需要至少15分钟的人在这项技术相当经验。因此, 有必要保持动物活着, 但在深麻醉下, 同时执行解剖, 以确保组织的可行性, 随后的电生理记录. 
 1. 灌注液和试验化合物的制备 <ol> <li> 准备 5 L 的克雷布斯溶液: 113 mm 氯化钠, 5.9 毫米氯化钾, 1.2 mm NaH 2 </sub...

<ol>
	<li>Al-Chaer, E. D., Traub, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+basis+of+visceral+pain:+recent+developments.">Biological basis of visceral pain: recent developments.</a> Pain. 96 (3), 221-225 (2002).</li><li>Christianson, J. A., Traub, R. J., Davis, B. 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A., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+recordings+of+afferent+fibres+with+receptive+fields+in+the+serosa,+muscle+and+mucosa+of+rat+colon.">In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon.</a> J Physiol. 518 (Pt 1), 271-282 (1999).</li><li>Page, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+contributions+of+ASIC+channels+1a,+2,+and+3+in+gastrointestinal+mechanosensory+function.">Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function.</a> Gut. 54 (10), 1408-1415 (2005).</li><li>Hockley, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2Y+Receptors+Sensitize+Mouse+and+Human+Colonic+Nociceptors.">P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.</a> J Neurosci. 36 (8), 2364-2376 (2016).</li><li>Peiris, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+visceral+afferent+recordings:+preliminary+report.">Human visceral afferent recordings: preliminary report.</a> Gut. 60 (2), 204-208 (2011).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum.">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300 (1), G170-G180 (2011).</li><li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents.">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33 (23), 9831-9839 (2013).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Functional+Characterization+of+Mouse+Colorectal+Afferent+Endings.">In vitro Functional Characterization of Mouse Colorectal Afferent Endings.</a> J Vis Exp. (95), e52310 (2015).</li><li>Nullens, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Recording+of+Mesenteric+Afferent+Nerve+Activity+in+Mouse+Jejunal+and+Colonic+Segments.">In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments.</a> J Vis Exp. (116), (2016).</li><li>Rong, W., Hillsley, K., Davis, J. B., Hicks, G., Winchester, W. J., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jejunalafferent+nerve+sensitivity+in+wild-type+and+TRPV1+knockout+mice.">Jejunalafferent nerve sensitivity in wild-type and TRPV1 knockout mice.</a> J Physiol. 560 (Pt 3), 867-881 (2004).</li><li>Brierley, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+chemosensory+function+and+receptor+expression+of+splanchnic+and+pelvic+colonic+afferents+in+mice).">Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice).</a> J Physiol. 567 (Pt 1), 267-281 (2005).</li><li>Brierley, S. M., Jones, R. C. 3. r. d., Gebhart, G. F., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice.">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127 (1), 166-178 (2004).</li><li>La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+expression+of+transient+receptor+potential+channel+V1,+transient+receptor+potential+channel+A1+and+mechanosensitive+two+pore-domain+K++channels+between+the+lumbar+splanchnic+and+pelvic+nerve+innervations+of+mouse+urinary+bladder+and+colon.">Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.</a> Neuroscience. 186, 179-187 (2001).</li><li>Wang, G., Tang, B., Traub, R. 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该协议是一个相对简单的实验方法来评估大鼠结肠传入的电生理特性。该协议 (从组织剥离到建立神经记录) 通常需要大约2小时才能完成。组织收集 (步骤 3) 和吸入电极 (步骤 5) 的制备是关键步骤。这是至关重要的, 能够找到 PG, LSN 和 PN, 并注意不要破坏神经节和神经组织解剖。玻璃吸管的尖端必须被打破和斜面到一个大小兼容的神经束。
该制剂通常是可行的几个小时, 允许研...

胃肠道 (GIT) 是丰富的支配与外部传入神经, 传达感官信号从肠道到中枢神经系统, 这有助于肠道-大脑的相互作用。改变兴奋性的这些外在传入, 以及改变中央处理的传入输入, 基础内脏疼痛和其他症状的胃肠条件, 包括功能性和炎症性肠道疾病1。来自直肠的感官信息主要通过胸腰椎/下和腰骶/盆腔神经 (PN)2传达。在啮齿动物疾病模型中, 对这些初级传入纤维的电生理特性的研究有了更大的兴趣。然而,在体内啮齿类动物结肠传入的电生理记录是一个技术挑战, 需要相当多的手术技巧。此外, 血液动力学的变化, 组织运动和麻醉药也可能影响神经活动和敏感性测试刺激在体内。因此, 近年来, 越来越多的研究采用了体外(前体内) 不同物种 (包括小鼠、大鼠、豚鼠和人类) 的制剂, 来研究结肠的感觉传导机制。传入和改变的兴奋性在疾病情况下。3,4,5,6,7,8
两种类型的前体结肠准备主要报告了: "平板" 准备5,9,10和 "管" 准备3,4。"平板" 小鼠直肠准备的视频协议以前已发布11。在本协议中, 小鼠直肠, 与 PN) 或腰椎内脏神经 (LSN) 附着, 是收获和 superfused 在一个组织室。直肠纵向切开, 将神经束延伸到充满石蜡油的记录室。使用单铂铱电极记录神经活动。该协议允许通过使用无偏电刺激来识别单个传入纤维的接受场。它本地化应用化学刺激, 以及应用不同的机械刺激范式 (例如,局灶性黏膜探查和周向伸展), 以传入神经末梢。因为神经必须从组织室延伸到一个单独的室, 所以保持附着的神经相对长是至关重要的;成功的神经解剖对这种方法的新的人构成挑战。最近, Nullens et al.发布了一个视频协议, 用于在小鼠空肠和结肠段 12中进行肠系膜传入的记录。在这个 "管" 的准备, 肠段与肠系膜附着保持完好, 从而允许分级扩张和 extra-luminal 管理不同的化学品。由于肠系膜神经被记录使用吸入电极, 它可以定位于组织附近, 即使肠系膜神经相对较短, 也可以记录传入活动。然而, 肠系膜神经包括迷走神经和脊髓传入纤维的混合种群, 支配空肠或胸腰椎下。腰骶部盆腔传入支配直肠, 不能在本协议中受到歧视。在这里, 我们提出了一个详细的协议, 对大鼠结肠传入的电生理记录使用 "管" 直肠制剂与完整的 PG。此方法可用于描述腰椎内脏 (下) 和腰骶盆腔传入的功能特性。

<table width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rats</td>
			<td>Any</td>
			<td>NA</td>
			<td>Any strain/sex can be used.</td>
		</tr>
	</tbody>
</table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

结肠感觉神经功能障碍已被牵连的病理生理学的几个常见的条件, 包括功能性和炎症性肠道疾病和糖尿病。在这里, 我们描述了一个协议的体外结肠传入在大鼠的电生理特性的表征。直肠, 与完整的盆腔神经节 (PG) 连接, 从鼠身上移除;superfused 与 carbogenated 克雷布斯溶液在记录室中;和中空的口腔和肛门两端允许扩张。从 PG 中产生的细神经束被识别, 并使用吸入电极记录多传入神经活动。结肠段的扩张引起多放电的逐渐增加。对低、high-threshold 和宽动态范围传入光纤进行了主成分分析。结肠传入的化学敏感性可通过对试验化合物的浴或腔内管理进行研究。本议定书可以修改, 以适用于其他物种, 如小鼠和豚鼠, 并研究不同的电生理特性的胸腰椎/下和腰骶/盆腔传入的下降结肠正常和病理条件。

图 1是为ex 体内"管" 直肠准备的实验性设置的示意图, 其中有代表从远端神经到 PG 的记录。神经可能包含骨盆和腰椎内脏传入的混合物。在正常大鼠的准备中, 结肠传入神经通常有不规则自发活动的低水平。结肠的斜坡扩张导致射击速度的逐渐增加 (图 1B)。通过绘制腔内压力-传入神经反应曲线 (图 1C...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

异常的感官功能是内脏疼痛和其他功能性和炎症性肠道疾病的症状。本文介绍了一种在前体大鼠直肠制备中结肠传入神经的电生理记录的协议。

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In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

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