这份手稿得到了美国国立卫生研究院的 R01DK118568 （MG）、R01DK124614 （MG） 和 R01HD105301 （MG）、Chan Zuckerberg Initiative Grant 2022-316749 （MG）、Thrasher 研究基金早期职业奖 （LCF）、UNC 儿童发展早期职业调查员资助 （LCF） 的支持，通过捐赠者对北卡罗来纳大学教堂山分校的慷慨支持， 以及北卡罗来纳大学教堂山分校的儿科系。

用于机制研究和药物发现的坏死性小肠结肠炎模型

<ol>
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作者声明与本手稿没有利益冲突。

这种 NEC-on-a-chip 系统是一种强大的新工具，可用于模拟 NEC 的病理生理学。该平台提供了一个复杂的微环境，通过结合具有连续管腔流动和拉伸的共培养系统，比以前的模型更接近 体内 肠道环境。这些条件促进了 3D 绒毛状结构的发展，该结构由高度极化的上皮组成，由成熟的上皮亚型和紧密连接组成（图 2）36。此外，顶端上皮表面很容易接触到实?...

坏死性小肠结肠炎 （NEC） 影响早产儿，出生体重 < 1500 g 的早产儿发病率高达 10%1。NEC 的病理生理学很复杂，包括肠上皮损伤、肠紧密连接破坏、肠道屏障通透性增加、免疫失调和上皮细胞死亡 2,3。我们对NEC发病机制的理解仍然不完整，尽管进行了数十年的研究，但仍然没有有效的靶向治疗。
推进NEC研究的一个重大障碍是从人类婴儿中分离出的原代肠道组织的可用性有限且尺寸小。从NEC患儿身上切除的肠道组织通常坏死并严重受损，这使得对疾病发病前机制的研究变得复杂。例如，患有NEC的婴儿的小肠被免疫细胞淹没，并且还观察到肠道干细胞数量减少，上皮细胞增殖减少和上皮细胞凋亡增加4,5,6,7。这导致从这些样品中培养肠上皮细胞以及分离RNA和蛋白质的困难，这些RNA和蛋白质可以在这种恶劣的炎症环境中降解。此外，由于手术NEC婴儿的疾病过程已经晚期，因此对诱发疾病的因素进行机制研究是不可行的。这些局限性导致依赖动物模型进行NEC的机理研究。
已经为小鼠、大鼠、仔猪、兔子和狒狒建立了NEC的动物模型5,8,9,11。动物模型的一个优势是，NEC样肠道疾病是由与人类NEC发病相关的因素诱发的，包括菌群失调的微生物组、反复缺氧发作和缺乏母乳喂养5,8,10,11。此外，在实验性NEC期间观察到的炎症反应和病理变化与人类疾病平行5,9,12。虽然这些模型模仿了人类NEC的许多特征，但NEC在动物和人类中的病理生理学之间存在固有的差异。例如，NEC的小鼠模型是在足月出生的小鼠中诱导的，尽管它们的肠道发育不完整，但NEC的病理生理学在这种临床背景下本质上是不同的。出生时小鼠肠道基因表达与存活前人类胎儿相似，直到第 14 天才接近妊娠 22-24 周的早产新生儿 （P14）13。这混淆了小鼠NEC模型，因为在P10之后通常不能在小鼠中诱导肠道损伤。此外，小鼠的近交系缺乏人类新生儿的免疫学14 和微生物多样性15，这是另一个混杂因素。因此，在NEC研究中增加将原发性人类样本纳入可提高该领域研究的临床相关性。
传统上，对 NEC 体外机制的研究利用了源自成人肠道癌细胞的单型细胞系，例如结直肠腺癌 （Caco2） 和人结肠腺癌 （HT-29） 细胞16。这些模型很方便，但由于它们从成体癌细胞生长、非极化结构以及与培养物重复传代相关的表型变化，因此生理相关性有限。肠样蛋白改进了这些模型，因为它们可以从肠组织的隐窝生长，分化为所有肠上皮亚型，并形成三维 （3D） 绒毛状结构17,18,19,20。最近，肠样蛋白与微流控技术相结合，开发了一种小肠芯片模型，并提供了更具生理相关性的体外模型系统21。
最初的器官芯片微流控设备是在 2000 年代初期推出的22,23,24。第一个器官芯片模型是人类呼吸肺芯片25。紧随其后的是许多单器官模型，如肠 21、肝脏 26、肾脏27、骨髓 28、血脑屏障 29 和心脏30。这些器官芯片模型已用于研究急性、慢性和罕见疾病，包括急性放射综合征、31 慢性阻塞性肺病、32 和神经退行性疾病 33。这些芯片上细胞的极化性质以及由多孔膜隔开的两个细胞区室的存在允许对复杂的生理过程进行建模，例如灌注、化学浓度梯度和免疫细胞趋化性34,35。因此，这些微流控系统为研究人类疾病的病理生理学和机制提供了新的工具。
Kasendra 等人于 2018 年描述了小肠芯片模型，他们利用儿科（10-14 岁）小肠活检标本分化成肠样并在微流控设备上培养21。血管内皮细胞、连续介质流动和拉伸/松弛也被纳入该模型。他们观察到肠上皮亚型分化、3D 绒毛状轴的形成、粘液产生和小肠基因表达模式21。随着 NEC-on-a-chip 系统的开发，该微流控模型被应用于新生儿疾病，该系统结合了新生儿肠样蛋白、内皮细胞和来自 NEC36 新生儿的微生物组。NEC-on-a-chip 概括了人类 NEC 的许多关键特征，包括炎症基因表达、特化上皮细胞的丧失和肠道屏障功能降低36。因此，该模型在NEC研究中有许多应用，包括机理研究和药物发现。在这篇手稿中，提供了NEC-on-a-chip模型性能的详细协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Leu15]-Gastrin I human</td>
			<td>Sigma-Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free (50x)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Bio-kit</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy 2 Multi-Mode Microplate Reader</td>
			<td>Agilent</td>
			<td>&#160;7131000</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND Methacrylate (PMMA) Cuvettes, Semi-Micro</td>
			<td>BrandTech</td>
			<td>759085D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354270</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Opus Real-Time PCR Systems</td>
			<td>Bio-Rad</td>
			<td>12011319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip Cradle</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip-S1 Stretchable Chip</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Sigma-Aldrich</td>
			<td>SML1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear TC-treated Multiple Well Plates,&#160; 48 well&#160;</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from human placenta</td>
			<td>Sigma-Aldrich</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I, powder</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Human Endothelial Cell Medium with Kit&#160;</td>
			<td>Cell Biologics</td>
			<td>H-1168</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Polypropylene Centrifuge Tubes, 50mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen&#160;</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II automated cell counter</td>
			<td>Invitrogen&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich</td>
			<td>D5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Cascade Blue, 3000 MW, Anionic, Lysine Fixable</td>
			<td>Invitrogen</td>
			<td>&#160;D7132</td>
			<td>Permeability dye&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable PES Filter Units, 0.2um aPES membrane</td>
			<td>Fisher Scientific</td>
			<td>FB12566504</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M,&#160; pH 8.0</td>
			<td>Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>ER-1 surface activation reagent</td>
			<td>Emulate</td>
			<td>ER-1</td>
			<td>Chip Activation Reagent 1</td>
		</tr>
		<tr>
			<td>ER-2 surface activation reagent&#160;</td>
			<td>Emulate</td>
			<td>ER-2</td>
			<td>Chip Activation Reagent 2</td>
		</tr>
		<tr>
			<td>Fibronectin Human Protein, Plasma</td>
			<td>Gibco</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Petri Dishes with Clear Lid, 100mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin-Based Coating Solution&#160;</td>
			<td>Cell Biologics</td>
			<td>6950</td>
			<td></td>
		</tr>
		<tr>
			<td>Genie Temp-Shaker 300</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-G300</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin&#160;</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, Liquid 1M Solution (238.3 mg/ mL)</td>
			<td>Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate&#160;</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Collagen Type I</td>
			<td>Sigma-Aldrich</td>
			<td>CC050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Primary Small Intestinal Microvascular Endothelial Cells</td>
			<td>Cell Biologics</td>
			<td>H-6054</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Fisher Scientific</td>
			<td>03-000-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp General Purpose Deluxe Water Baths</td>
			<td>Fisher Scientific</td>
			<td>FSGPD10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB) agar, Miller</td>
			<td>Supelco</td>
			<td>L3027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN Cells&#160;</td>
			<td>American Type Culture Collection</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Basement Membrane Matrix, LDEV-free&#160;</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Cell Culture Matrix</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>1009005</td>
			<td></td>
		</tr>
		<tr>
			<td>NAILSTAR UV LAMP</td>
			<td>NailStar</td>
			<td>NS-01-US</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC&#160;Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-274200</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>72340</td>
			<td></td>
		</tr>
		<tr>
			<td>Orb-HM1 Hub Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>ThermoFisher</td>
			<td>047392.9L</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L8-200XLS+</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips TR LTS 1000&#181;L S 768A/8</td>
			<td>Rainin</td>
			<td>17014966</td>
			<td></td>
		</tr>
		<tr>
			<td>Pod Portable Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Grade Fetal Bovine Serum (FBS)(Heat Inactivated)&#160;</td>
			<td>Avantor Seradigm</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Reverse Transcription Kit&#160;</td>
			<td>QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SB 431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>Square BioAssay Dish with Handles, not TC-treated&#160;</td>
			<td>Corning</td>
			<td>431111</td>
			<td></td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR&#160;Green Supermix</td>
			<td>Bio-Rad</td>
			<td>1725271</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GV Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers, 70um</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringes, 10mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-453</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight, fine, sharp point scissors</td>
			<td>Miltex&#160;Instruments</td>
			<td>MH5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Sorvall X4R Pro-MD Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75016052</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>TRIzol Reagent&#160;</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>RNA extraction reagent</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4% (w/v) in PBS, pH 7.5 &#177; 0.5</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>Enzymatic Dissociation Reagent</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>VIOS 160i CO2 Incubator, 165 L</td>
			<td>Thermo Scientific</td>
			<td>13-998-252</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Zo&#235;-CM1 Culture Module</td>
			<td>Emulate</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic model of necrotizing enterocolitis incorporating human neonatal intestinal enteroids and a dysbiotic microbiome

坏死性小肠结肠炎 （NEC） 是一种严重且可能致命的肠道疾病，由于其复杂的发病机制而难以研究，目前尚不完全清楚。NEC 的病理生理学包括肠道紧密连接破坏、肠道屏障通透性增加、上皮细胞死亡、微生物菌群失调和炎症失调。研究NEC的传统工具包括动物模型、细胞系以及人类或小鼠肠道类器官。虽然使用这些模型系统的研究提高了该领域对疾病病理生理学的理解，但它们概括人类NEC复杂性的能力是有限的。现在已经开发了一种使用微流控技术的改进的NEC 体外 模型，称为NEC-on-a-chip。NEC-on-a-chip 模型由一个微流控装置组成，该装置接种了源自早产新生儿的肠样蛋白，与人内皮细胞和来自患有严重 NEC 的婴儿的微生物组共培养。该模型是NEC病理生理学机制研究的宝贵工具，也是新生儿肠道疾病药物发现测试的新资源。在本手稿中，将提供 NEC-on-a-chip 模型的详细说明。

将肠样体接种到微流控装置上（图1）并如上所述培养。通过明场显微镜监测接种前细胞培养基质水凝胶中肠样蛋白的生长，然后在接种装置后随后的肠上皮细胞单层扩增（图2）。形成汇合的肠上皮细胞单层，随后发育成成熟的 3D 绒毛状结构（图 2）。这种接种有肠源性新生儿肠上皮和 HIMEC 的微流控装置被称为新生儿肠芯片模型<...

Watch this Scientific Journal Video about Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model at JoVE.com

作者聚焦：使用 NEC-on-a-Chip 模型增强理解和治疗策略 

该方案描述了坏死性小肠结肠炎（NEC）的 体外 模型，可用于疾病发病机制的机制研究。它的特点是微流控芯片，其中植入了源自人类新生儿肠道、内皮细胞和患有严重 NEC 的新生儿肠道微生物组的肠道类生物。

结合人新生儿肠道类生物和菌群失调微生物组的坏死性小肠结肠炎微流控模型

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

该领域的研究具有挑战性，因为坏死性小肠结肠炎的病理生理学很复杂，而这种复杂性并没有反映在许多常用的体外模型中。使用人体样本的研究通常受到样本量小和肠道手术时获得的样本稀有性的限制。我们的目标是确定导致早产儿发生NEC的机制，并确定新的治疗策略。NEC-on-a-Chip系统通过更紧密地反映新生儿肠道的生理机能，改进了以前的模型。在这个模型中，我们将患者来源的肠道类器官、内皮细胞和来自患有严重 NEC 的新生儿的微生物组结合到微流控芯片上。

microfluidic-model-necrotizing-enterocolitis-incorporating-human

Research

JoVE Journal

Medicine

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

1.1K 次观看.  University of North Carolina at Chapel Hill. 该领域的研究具有挑战性，因为坏死性小肠结肠炎的病理生理学很复杂，而这种复杂性并没有反映在许多常用的体外模型中。使用人体样本的研究通常受到样本量小和肠道手术时获得的样本稀有性的限制。我们的目标是确定导致早产儿发生NEC的机制，并确定新的治疗策略。NEC-on-a-Chip系统通过更紧密地反映新生儿肠道的生理机能，改进了以前的模型。在这个模型中，我们...

视频: 作者聚焦：使用 NEC-on-a-Chip 模型增强理解和治疗策略 

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生儿窒息了猪模型

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

科研

医学

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.