Name: ex vivo hepatic perfusion through the portal vein in mouse
Uploaded: 2022-03-09T12:30:00
Description: Watch this Scientific Journal Video about Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse at JoVE.com

这项工作得到了美国国立卫生研究院（R01-DK105346，P41-GM122698，5U2C-DK119889）的资助。这项工作的一部分是在国家高磁场实验室的高级磁共振成像和光谱（AMRIS）设施的麦克奈特脑研究所进行的，该设施得到了国家科学基金会合作协议No的支持。DMR-1644779和佛罗里达州。

涉及小鼠的实验按照佛罗里达大学机构动物护理和使用委员会（协议编号#201909320）进行处理。使用的小鼠菌株为C57BL / 6J;所有小鼠都是雄性。这种方法通常也适用于使用其他标准小鼠品系的研究。该手术由两个人一起工作进行。
1. 初始设置
<ol><li>用含有克雷布斯 - 亨赛莱特电解质6（25 mM NaHCO3，112 mM NaCl，4.7 mM KCl，MgSO 4，KH 2<sub...

<ol>
	<li>Ragavan, M., McLeod, M. A., Giacalone, A. G., Merritt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+Dihydroxyacetone+Is+a+Sensitive+Probe+of+Hepatic+Gluconeogenic+State.">Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State.</a> Metabolites. 11 (7), 441 (2021).</li><li>Lumata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+(13)C+Magnetic+Resonance+and+Its+Use+in+Metabolic+Assessment+of+Cultured+Cells+and+Perfused+Organs.">Hyperpolarized (13)C Magnetic Resonance and Its Use in Metabolic Assessment of Cultured Cells and Perfused Organs.</a> Methods in Enzymology. 561, 561-573 (2015).</li><li>Moreno, K. X., 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=Real-time+detection+of+hepatic+gluconeogenic+and+glycogenolytic+states+using+hyperpolarized+[2-13C]+dihydroxyacetone.">Real-time detection of hepatic gluconeogenic and glycogenolytic states using hyperpolarized [2-13C] dihydroxyacetone.</a> The Journal of Biological Chemistry. 289 (52), 35859-35867 (2014).</li><li>Moreno, K. X., 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=Hyperpolarized+&#948;-[1-13C]+gluconolactone+as+a+probe+of+the+pentose+phosphate+pathway.">Hyperpolarized &#948;-[1-13C] gluconolactone as a probe of the pentose phosphate pathway.</a> NMR in Biomedicine. 30 (6), (2017).</li><li>Merritt, M. E., Harrison, C., Sherry, A. D., Malloy, C. R., Burgess, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flux+through+hepatic+pyruvate+carboxylase+and+phosphoenolpyruvate+carboxykinase+detected+by+hyperpolarized+13C+magnetic+resonance.">Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (47), 19084-19089 (2011).</li><li>Bailey, L. E., Ong, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krebs-Henseleit+solution+as+a+physiological+buffer+in+perfused+and+superfused+preparations.">Krebs-Henseleit solution as a physiological buffer in perfused and superfused preparations.</a> Journal of Pharmacological Methods. 1 (2), 171-175 (1978).</li><li>Kolwicz, S. C., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Cardiac+Function+and+Energetics+in+Isolated+Mouse+Hearts+Using+31P+NMR+Spectroscopy.">Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy.</a> Journal of Visualized Experiments: JoVE. (42), e2069 (2010).</li><li>Hwang, G. H., 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=Protective+effect+of+butylated+hydroxylanisole+against+hydrogen+peroxide-induced+apoptosis+in+primary+cultured+mouse+hepatocytes.">Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes.</a> Journal of Veterinary Science. 16 (1), 17-23 (2015).</li><li>Bessems, 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=The+isolated+perfused+rat+liver:+standardization+of+a+time-honoured+model.">The isolated perfused rat liver: standardization of a time-honoured model.</a> Laboratory Animals. 40 (3), 236-246 (2006).</li><li>Beal, E. W., 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=A+Small+Animal+Model+of+Ex+Vivo+Normothermic+Liver+Perfusion.">A Small Animal Model of Ex Vivo Normothermic Liver Perfusion.</a> Journal of Visualized Experiments: JoVE. (136), e57541 (2018).</li><li>Collins, J. B., Song, J., Mahabir, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+and+duration+of+intradermal+mixtures+of+bupivacaine+and+lidocaine+with+epinephrine.">Onset and duration of intradermal mixtures of bupivacaine and lidocaine with epinephrine.</a> The Canadian Journal of Plastic Surgery. 21 (1), 51-53 (2013).</li><li>. Medical Dictionary Available from: <a target="_blank" href="https://www.merriam-webster.com/medical">https://www.merriam-webster.com/medical</a> (2022)</li><li>Thorpe, D. 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Heparin Available from: <a target="_blank" href="https://go.drugbank.com/drugs/DB01109">https://go.drugbank.com/drugs/DB01109</a> (2022)</li><li>Overmyer, K. A., Thonusin, C., Qi, N. R., Burant, C. F., Evans, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+and+euthanasia+on+metabolomics+of+mammalian+tissues:+studies+in+a+C57BL/6J+mouse+model.">Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model.</a> PloS One. 10 (2), 0117232 (2015).</li></ol>

这种外科手术具有挑战性，需要大量的实践才能获得可重复的结果。应根据需要调整异氟醚和载气，以通过尽可能多的外科手术来维持动物的生存能力。环境，一天中的时间，年龄，体重和其他几个因素都会影响麻醉。体重，饮食，小鼠品系和年龄可能会影响手术，因为脂肪堆积会干扰门静脉的可视化。当用胶带将爪子贴下来时，必须注意不要对颈部施加任何可能导致窒息的压力。此外，小鼠越?...

离体灌注在肝脏代谢研究中至关重要，通过门静脉灌注是这些研究的标准。为了单独研究肝脏代谢，必须从体内切除肝脏，以避免其他器官代谢引起的并发症（即全身代谢），并控制激素的可用性（胰岛素，胰高血糖素等）。这种方法对于了解糖尿病，NAFLD和NASH等疾病对肝脏代谢的影响以及药物作用机制至关重要。本文可作为肝切除和灌注的指南。我们开发了一种简化的程序，以足够的严谨性和可重复性进行这些代谢肝脏研究。如果手术不正确，则获得的代谢数据存在显着的变异性。我们描述了一种在核磁共振（NMR）光谱仪中原位代谢研究的背景下进行门静脉导管插入术和肝切除的有组织方法，如文献1，2，3，4，5中所述。
目前，没有文献描述在 NMR 内使用玻璃柱进行 离体 肝灌注。也没有视频或文本出版物提供如何使用小鼠肝脏进行手术的清晰示例，特别是演示如何插入门静脉，切除肝脏，转移和将肝脏悬挂在玻璃柱上。由于转基因小鼠无处不在地用于研究肝脏代谢，这是一个值得完整描述的基本程序。肝灌注手术并不新鲜，但本文是一种黄金标准方法，并附有一段视频，演示本文中描述的技术卓越性，以帮助所有对此程序感兴趣的人。这里介绍的方法最好应用于实时代谢，以检测疾病模型中代谢物的功能和周转。
该方法使用100厘米的水套玻璃柱，允许肝脏悬挂在由灌注液封装在NMR管内的套管底部。玻璃夹套中的热水用于控制灌注液温度。薄层氧合器用95%/5%O2/CO2 加压以控制pH值。通过使用三个单独的泵，可以设置灌注液柱高度，从而为肝脏提供恒定的压力。在施加恒定压力之外，流量不受控制（图1）。为了确认肝脏功能正常，氧气测量与流速一起进行。在我们手中，这组先决条件导致高度可重复的NMR实验，用于评估肝脏代谢功能。

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		<tr>
			<td>1 mL Luer-Lock Single Use Sterile Disposable Syringe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Non-specific Brand</td>
		</tr>
		<tr>
			<td>100 cm Water Jacketed Glass Column</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom Made</td>
		</tr>
		<tr>
			<td>2-0 Silk Suture</td>
			<td>Braintree Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>22 Gauge Catherter 1 in. Without Safety</td>
			<td>Terumo</td>
			<td>SRFF2225</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G 0.75 in. Hypodemeric Needles</td>
			<td>Exel International</td>
			<td>26407</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G 1.5 in. Hypodemeric Needles</td>
			<td>Exel International</td>
			<td>26426</td>
			<td></td>
		</tr>
		<tr>
			<td>4x4 in. Surgical Platform</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom Made</td>
		</tr>
		<tr>
			<td>70% Alcohol Wipe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Non-specific Brand</td>
		</tr>
		<tr>
			<td>Circulating Water Bath</td>
			<td>MS Lauda</td>
			<td>N/A</td>
			<td>Model no longer manufactured</td>
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		<tr>
			<td>Cotton Tip Applicator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Non-specific Brand</td>
		</tr>
		<tr>
			<td>Delicate Operating Scissors; Straight; Sharp-Sharp; 30mm Blade Length; 4 3/4 &#34;</td>
			<td>Roboz</td>
			<td>RS-6702</td>
			<td></td>
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			<td>Dumont #5/45 Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11251-35</td>
			<td></td>
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			<td>Dumont #7 - Fine Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11274-20</td>
			<td></td>
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			<td>Hemostats</td>
			<td>Fine Scientific Tools</td>
			<td>13015-14</td>
			<td></td>
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			<td>Heparin Sodium Injectable 1000 units/mL</td>
			<td>RX Generics</td>
			<td>71288-0402-02</td>
			<td></td>
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		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-0704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine HCl 2%</td>
			<td>VEDCO Inc.</td>
			<td>50989-0417-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane-Thin-Layer Oxygenator</td>
			<td>Radnoti</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors; Curved; Blunt; 27 mm Blade Length; 5 &#34;</td>
			<td>Roboz</td>
			<td>RS-6013</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Meter System</td>
			<td>Hanstech Instruments Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline 0.9% Solution</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Saline is made in lab</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Non-specific Brand</td>
		</tr>
		<tr>
			<td>&#160;Variable Speed Analog Console Pump Systems</td>
			<td>Cole Palmer</td>
			<td>N/A</td>
			<td>Models are custom per application</td>
		</tr>
		<tr>
			<td>Weigh boats</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Non-specific Brand</td>
		</tr>
	</tbody>
</table>

ex vivo hepatic perfusion through the portal vein in mouse

糖尿病、糖尿病前期、非酒精性脂肪性肝病 （NAFLD） 和非酒精性脂肪性肝炎 （NASH） 等代谢性疾病正变得越来越普遍。离体肝灌注允许在可以严格控制的营养条件下使用核磁共振（NMR）对肝脏代谢进行全面分析。由于计算机模拟仍然是评估激素作用和药物干预效果的主要理论手段，因此灌注肝脏仍然是了解肝脏代谢的最有价值的测试台之一。由于这些研究指导了对肝脏生理学的基本见解，因此结果必须准确且可重复。离体肝灌注再现性的最大因素是手术质量。因此，我们引入了一种有组织和简化的方法，用于在原位NMR实验的背景下进行离体小鼠肝脏灌注。我们还描述了一种独特的应用，并讨论了在这些研究中遇到的常见问题。总体目的是为我们多年来改进的技术提供一个简单的指导，我们认为这是在原位NMR实验中获得肝切除和灌注可重复结果的黄金标准。磁体到磁场中心的距离以及组织在NMR实验期间无法进行干预使我们的方法新颖。

肝功能主要通过耗氧量和流速来评估。典型流速为 4-8 mL/min，耗氧量为 1 μmol/min.g。这些措施将根据特定的实验条件和生物学差异而有所不同。
使用异氟醚的确切量将取决于所使用的麻醉系统的类型以及小鼠的环境和年龄/体重。在手术过程中，异氟醚和输送气体不会改变，尽管根据手术区域的具体情况（例如，背景噪音）可能需要一些变化10。当肝素深部皮?...

Watch this Scientific Journal Video about Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse at JoVE.com

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

该协议描述了一种通过门静脉灌注切除完整小鼠肝脏以进行代谢研究的简单方法。

离体 小鼠通过门静脉进行肝灌注

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

该视频演示了一种简化的程序，用于进行具有足够严谨性和可重复性的代谢肝脏研究，可以作为进行肝切除术以进行体内灌注研究的指南。该技术最好应用于实时代谢以检测功能，在疾病模型中的周转代谢物中。灌注可以精确控制肝脏可用的营养物质和激素。虽然该技术不针对治疗，但肝脏灌注有助于了解疾病（如糖尿病、NAFLD 和 NASH）对肝脏代谢的病因和影响。随着新药的开发，评估肝脏中枢能量代谢的方法变得至关重要。这种方法可用于几种疾病的模型，包括癌症和疾病进展的几个不同阶段。首先，在麻醉诱导后将小鼠的鼻子放入鼻锥中，然后贴下爪子。利多卡因通过皮下注射在髂前嵴区域进行双侧注射。进行脚趾捏合测试以确认不存在所有疼痛反射。进行腹腔切开术，通过做一个三厘米宽的切口来暴露内脏器官。使用止血剂扩大切口，止血剂通过夹紧 xiphoid 过程来拉动牵引力。使用棉尖涂药器清除覆盖门静脉的小肠和大肠。将丝状缝线放置在门静脉弓下，靠近肝脏。使用2-0缝线将第二丝缝线放在离肝脏远端的肠系膜下静脉的近端或远端。缝合线就位后，用22号导管对门静脉进行插管，使斜面保持朝上。以不超过 15 度角进入门静脉。将第一根缝线绑过导管穿刺器。门静脉插管后，用丝缝线将导管从门静脉分支远端两到三毫米固定。用第二根缝线固定导管的下部。用缝合线打结，将导管固定在门静脉的远端部分和周围组织。固定导管后，将一个1毫升注射器与38.1毫米长度的27号针头插入导管中以冲洗血液和气泡。使用一毫米内径和五毫米外径硅胶管，带有固定的止动旋塞，将灌注柱连接到导管上，允许缓冲液流入肝脏，标志着灌注的开始。启动计时器后，使用剪刀切入下腔静脉以缓解增加的血管压力。通过观察肝脏颜色从粉红色或红色到淡黄色的均匀变化，确认灌注物通过肝脏的流动。一旦确认血流，切除胃、小肠、大肠和周围组织的右肾。在手术助理的帮助下，当外科医生穿过顶叶腹膜和胸组织切除肝脏时，在腹腔和胸腔周围移动肝脏。向上抬起肝脏，用剪刀切开剩余的结缔组织，将肝脏固定到位。慢慢操纵肝脏，以便于观察。流速和耗氧量测量对于监测肝脏的健康和功能至关重要。喂养和禁食肝脏之间的耗氧量通常略有不同，这可归因于禁食肝脏中糖发生引起的能量需求增加。最重要的步骤包括以15度角插入导管，观察导管尖端，系紧缝线时滚动手腕和肩部，以及避免门静脉扭转。灌注后，可以使用任意数量的生化或分析方法研究切除的组织。离体灌注与实时MRI和光谱学相结合，可以实时测量代谢通量。当然，这延伸到对疾病的反应而从控制状态对通量的扰动。

Research

JoVE Journal

Biochemistry

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

脑膜分离法<em&gt;体检</em评估亚突触蛋白定位的方法

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

离体 小鼠通过门静脉进行肝灌注

离体小鼠通过门静脉进行肝灌注