作者感谢圣保罗大学医学诊所的毕业后计划;Coordenação de Aperfeiçoamento de Pessoal de Nível Superior（CAPES）和圣保罗大学医学院（FMUSP）。

该协议已获得USP医学院动物使用伦理委员会的批准，编号项目：1343/2019-CEUA：FMUSP。对于该协议，使用C57BL / 6小鼠，年龄为6周，体重20±2g（n = 9 /组）。
1. 剖腹手术
<ol><li>使用1 mL注射器和13x0.45mm针头26G 1/2皮下注射（0.1 mL / 10g体重）用木精（10mg / kg）和氯胺酮溶液（80mg / kg）麻醉动物。通过捏住脚趾检查是否有足够的麻醉深度。使用加热垫控制体温。确保所有手术材料都是无?...

<ol>
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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=Review+of+experimental+animal+models+of+biliary+acute+pancreatitis+and+recent+advances+in+basic+research.">Review of experimental animal models of biliary acute pancreatitis and recent advances in basic research.</a> HPB (Oxford). 14 (2), 73-81 (2012).</li><li>Mayerle, J., Sendler, M., Lerch, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretagogue+(Caerulein)+induced+pancreatitis+in+rodents.">Secretagogue (Caerulein) induced pancreatitis in rodents.</a> Pancreapedia: The Exocrine Pancreas Knowledge Base. 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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=Anti-inflammatory+effects+of+peritoneal+lavage+in+acute+pancreatitis.">Anti-inflammatory effects of peritoneal lavage in acute pancreatitis.</a> Pancreas. 39 (8), 1180-1184 (2010).</li><li>Perides, G., Acker, G. J. v., Laukkarinen, J. M., Steer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+acute+biliary+pancreatitis+induced+by+retrograde+infusion+of+bile+acids+into+the+mouse+pancreatic+duct.">Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.</a> Nature Protocols. 5 (2), 335-341 (2010).</li><li>Wittel, U. A., 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=Taurocholate-induced+pancreatitis:+a+model+of+severe+necrotizing+pancreatitis+in+mice.">Taurocholate-induced pancreatitis: a model of severe necrotizing pancreatitis in mice.</a> Pancreas. 36 (2), 9-21 (2008).</li><li>Tao, L., Reese, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+mouse+models+that+reflect+human+immune+responses.">Making mouse models that reflect human immune responses.</a> Trends Immunology. 38 (3), 181-193 (2017).</li><li>Vandamme, T. 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(1), (2010).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+peritoneal+cavity+cells.">Isolation of mouse peritoneal cavity cells.</a> Journal of Visualized Experiments. (35), e1488 (2010).</li><li>Schmidt, 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=A+better+model+of+acute+pancreatitis+for+evaluating+therapy.">A better model of acute pancreatitis for evaluating therapy.</a> Annals of Surgery. 215 (1), 44-56 (1992).</li><li>Liu, D. L., 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=Resveratrol+improves+the+therapeutic+efficacy+of+bone+marrow-derived+mesenchymal+stem+cells+in+rats+with+severe+acute+pancreatitis.">Resveratrol improves the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in rats with severe acute pancreatitis.</a> International Immunopharmacology. 80, 106128 (2020).</li><li>Yang, X. F., 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=Chaiqin+chengqi+decoction+alleviates+severe+acute+pancreatitis+associated+acute+kidney+injury+by+inhibiting+endoplasmic+reticulum+stress+and+subsequent+apoptosis.">Chaiqin chengqi decoction alleviates severe acute pancreatitis associated acute kidney injury by inhibiting endoplasmic reticulum stress and subsequent apoptosis.</a> Biomedicine &amp; Pharmacotherapy. 125 (12), 110024 (2020).</li><li>Yang, X. F., 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=Chaiqin+chengqi+decoction+alleviates+severe+acute+pancreatitis+associated+acute+kidney+injury+by+inhibiting+endoplasmic+reticulum+stress+and+subsequent+apoptosis.">Chaiqin chengqi decoction alleviates severe acute pancreatitis associated acute kidney injury by inhibiting endoplasmic reticulum stress and subsequent apoptosis.</a> Biomedicine &amp; Pharmacotherapy. 125, 110024 (2020).</li><li>Venglovecz, V., Z, R., Hegyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+bile+acids+on+pancreatic+ductal+cells.">The effects of bile acids on pancreatic ductal cells.</a> Pancreapedia: The Exocrine Pancreas Knowledge Base. (1), (2019).</li><li>Roberts, S. E., Akbari, A., Thorne, K., Atkinson, M., Evans, P. 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通过逆行牛磺胆酸钠输注诱导急性胰腺炎的方法已经在大鼠中显示22，23，24。分别于2008年、2010年和2015年出版的三部类似作品作为议定书的参考3，14，15。在这项工作中，我们列出了在C57BL / 6小鼠中重现该方法的所有关键步骤以及验证它的一些可能性。
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在人类中，胆结石的存在是胰腺炎的最常见原因，这是由于胆总管末端部分的阻塞，中断了胰腺分泌物的流动并导致胰腺中强烈的炎症过程，血清和炎症介质中消化酶的浓度增加1，2。
已经提出了两种不同的理论来解释急性胰腺炎（AP）的发展。"共通道"理论表明，胆囊中存在的结石会阻塞远端胆总管系统，从而使胆汁分泌逆行流入胰管。第二种理论（"导管阻塞"理论）表明，过量胆结石阻塞胰管会导致胰腺分泌物流向十二指肠的阻塞，从而导致导管高血压3。虽然导致急性胆道胰腺炎的机制尚不完全清楚，但结果是强烈的炎症过程。消化酶出疹和胰腺自身消化导致组织病理学改变，腹水和血清中炎症细胞因子（IL-1β，IL-6，TNF-α）增加，以及急性期蛋白增加4，5，6。
严重的急性胰腺炎是一种值得临床关注的疾病，因为它涉及多个器官和高死亡风险。用于急性胰腺炎（AP）繁殖的动物模型很重要，因为它们解释了疾病的病理生理机制，并有助于监测炎症事件的演变，从疾病的初始阶段开始。这在诊所中通常是不可能的2，7。此外，在临床前研究中，胰腺组织的获取很容易，有利于阐明与临床条件相关的变化8 以及使用等基因物种的可能性，消除不良变量，并反映临床与人类状况中观察到的结果的相似性9。
用于诱导大鼠和小鼠急性胰腺炎的胆道和非胆道模型已在科学文献中频繁研究。非胆道诱导方法包括给予超近端刺激剂量的胆囊收缩素促分泌剂或其类似物cerulein10;给予几乎致命剂量的l-精氨酸;或给予补充乙硫氨酸的胆碱缺乏饮食11。虽然这些方法很容易复制并导致胰腺炎症，但它们不会复制理论上触发AP的机制（即胆汁分泌物反流进入胰管）。解决胆道模型的技术是基于胆汁酸逆行输注到胰管中，需要训练有素的研究人员来执行该协议。已经发表了几项在大鼠中使用这种方法的研究（显然是出于技术原因，因为这些实验涉及外科手术）12，13。然而，在小鼠中的方法可能会在炎症研究中提供更有趣的结果3，14，15。在这项研究中，我们将展示通过输注牛磺胆酸钠在C57BL / 6麻醉小鼠中繁殖严重急性胰腺炎的步骤清单。
对于涉及需要抗体实验以及基因和蛋白质表达分析的工作，使用小鼠是优选的，因为这些动物的材料库更多，并且有可能使用等基因和敲除物种，以及其他可用于研究16。小鼠C57BL / 6是一种小鼠近交系，最初用于研究抗肿瘤活性和免疫学。该菌株越来越被研究人员视为同种异基因，允许结果具有更大的可重复性，这可能意味着在实验中使用较少数量的动物，并且同一组之间结果的变异性较小17，18。
Perides等人（2010）14 发表了一种通过牛磺胆酸钠输注在小鼠中诱导AP的方案。在这里，我们使用C57BL / 6小鼠中更高的牛磺胆酸钠浓度（2.5%）更新该模型，并具有定义的输注体积和速度（图1）。在小鼠诱导后12小时内达到最大严重程度。血清和腹膜腔中IL-6浓度的升高与AP的进展相关。通过实践，从麻醉诱导到输注完成的总估计时间为每只动物25分钟。训练有素的研究人员进行这项实验至关重要。为确保将溶液正确注射到胆总管中，请使用亚甲蓝而不是牛磺胆酸钠进行几次飞行员培训课程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm needle</td>
			<td>INTRAG MEDICAL TECH</td>
			<td>90183210</td>
			<td>30G</td>
		</tr>
		<tr>
			<td>&#160;0.54 mm polyethylene tube</td>
			<td>Tygon</td>
			<td>730010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Styrofoam block</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>masking tape for mounting the mouse</td>
			<td>Missner</td>
			<td>1236</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Infusion pump scheduled to 10&#181;L / min.</td>
			<td>Havard aparatus-Peristaltic Pump Series</td>
			<td>MA1 55-7766</td>
			<td>&#160;Model 66 Small Peristaltic</td>
		</tr>
		<tr>
			<td>Scissors and forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic providine iodine</td>
			<td>Pfizer</td>
			<td>12086OR</td>
			<td>antisepsis</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SIGMA</td>
			<td>459836</td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Lord</td>
			<td>bdk9a1ghk6</td>
			<td>For trichotomy</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td>86339- 1G</td>
			<td>CAS NUMBER- 345909-26-4</td>
		</tr>
		<tr>
			<td>microvessel clip</td>
			<td>Medicon Surgical</td>
			<td>56.87.35</td>
			<td>Approximator, opening 4.0 mm, closing pressure 30 -&#160;40 g</td>
		</tr>
		<tr>
			<td>6-0 prolene</td>
			<td>Bioline</td>
			<td>5162</td>
			<td>Suture line</td>
		</tr>
		<tr>
			<td>Ketamin NP (cloridrato de dextrocetamina) 50mg/mL</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xilazine 2%</td>
			<td>Syntec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline solution (0.9% (wt/vol) saline)</td>
			<td>Farmace</td>
			<td>105851</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Blue</td>
			<td>Sigma-Aldrich Chemicals</td>
			<td>M5528</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX&#160;MAP&#160;Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay</td>
			<td>MERCK</td>
			<td>MCYTOMAG-70K</td>
			<td>Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.</td>
		</tr>
		<tr>
			<td>Amylase Assay</td>
			<td>Labtest</td>
			<td>11</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmarres retractor 13-mm 
			width</td>
			<td>ROBOZ</td>
			<td>RS-6672</td>
			<td></td>
		</tr>
	</tbody>
</table>

sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice

通过牛磺胆酸钠输注诱导胆道急性胰腺炎已被科学界广泛使用，因为代表了人类临床状况和与临床胆道胰腺炎发作相对应的炎症事件的再现。胰腺损伤的严重程度可以通过测量输注胆汁酸的浓度、速度和体积来评估。本研究提供了方案生殖中使用的材料和方法的更新清单，并显示了该急性胰腺炎（AP）模型的主要结果。以前的大多数出版物都仅限于在大鼠中复制这种模型。我们已经将这种方法应用于小鼠，这提供了可能与研究相关的其他优势（即，这些动物的试剂和抗体库的可用性以及使用转基因小鼠品系的可能性）。对于小鼠急性胰腺炎诱导，我们提出了一个系统方案，在C57BL / 6小鼠中以10μL / min的输注速度以2.5%牛磺胆酸钠的定义剂量3分钟，在诱导后12小时内达到其最大严重程度，并突出结果并具有验证该方法的结果。通过实践和技术，从麻醉诱导到输注完成的总估计时间为每只动物25分钟。

根据施密特量表21 ，胰腺炎严重程度的评分在0-3之间，其中0对应于不存在，1对应于轻度存在（<25%），2对应于中度存在（在25和50%之间），3对应于强烈的存在（>50%）（表1）。进行的测量是血浆淀粉酶活性，胰腺水肿，腺泡细胞，损伤/坏死，胰腺炎症（通过H&E染色切片的组织学分析）以及血清和PerC液体中的IL-6细胞因子浓度。严重AP12小时后?...

Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

严重急性胰腺炎的动物模型能够在初始阶段研究病理生理变化，促进观察炎症事件的演变。在这里，我们提供了通过将牛磺草酸钠逆行输注到麻醉的C57BL / 6小鼠的胰管中来诱导严重急性胆汁性胰腺炎的方案。

牛磺胆酸钠诱导C57BL / 6小鼠严重急性胰腺炎

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

该方法可用于重现急性胆道胰腺炎中发生的炎症事件。可以在早期阶段研究胰腺组织的变化，这在临床上通常是不可能的。这种技术的主要优点是，对于训练有素的研究人员来说，繁殖起来既快速又容易，并且它提供了与人类急性胰腺炎相似的结果。该方法可用于研究药物分子的药理学免疫反应和其他治疗干预措施，因为它允许研究胰腺和邻近器官中的炎症介导细胞。在尝试该协议时，定位器官并建立插管区域至关重要。记住器官的位置，并首先使用亚甲蓝等有色溶液进行注射应用。用脚趾捏检查麻醉深度后，用5%聚维酮碘溶液清洁麻醉小鼠的腹部区域。使用修剪器去除胸部和下腹部之间的毛发，并用70%的酒精清洁约两平方厘米的手术区域。将动物固定在手术板上，用剪刀水平切开腹部上部五毫米的皮肤，并在西腓手术下方一厘米处切割。在腹膜上重复切口，以最小的腔暴露进行剖腹手术。使用牵开器，将肝脏拉向头部，距离肠道约一厘米，并定位胰腺和十二指肠的区域。使用镊子，将肝脏抬向动物头部。通过轻轻拉动小肠的部分，用6-0聚丙烯缝合线固定两个侧端，以便更好地观察胆总管的远端。使用微血管夹，暂时闭塞近端胆总管。将器官暴露在腹腔外。然后用8-0暂时闭塞远端胆总管缝合。为了进入胆总管，刺穿壶腹周围区域，该区域表现为小肠壁的白色部分。将0.4毫米的针头连接到0.54毫米的聚乙烯管，启动输液泵，以每10克体重10微升的恒定速度输注2.5%牛磺胆酸钠溶液三分钟。输液后，取出微血管夹，临时8-0缝合线，并将注射针头从胰胆管中取出。用6-0非吸水性单丝聚丙烯缝合线缝合腹部。剖腹手术和末端缝合之间的时间不应超过30分钟。手术后，将动物放在聚乙烯箱中，里面衬有木屑和水，随意食物。轻轻握住动物，将皮肤向后拉，促进眼球的轻微突出，并将动物的眼睛朝上放置。在动物的眼睛中滴入含有局部麻醉剂的眼药膏。将毛细管的末端置于眼角，并以约30至45度的角度将其轻轻插入眼球下方。旋转毛细管直至血流开始。收集结束后，通过轻轻按压眼睑来确保体内平衡。之后，使用27号针，将四毫升冰冷的PBS注射到小鼠的腹膜腔中。注射后，轻轻按摩腹膜10秒钟以去除粘附的细胞。使用剪刀和镊子，在内层皮肤和肌肉组织上做一小块0.5厘米的切口，露出腹腔。将球形移液管插入腹膜并收集尽可能多的液体，注意不要吸入脂肪组织。从头部区域收集一部分胰腺进行进一步处理。将收集的液体沉积在冰上保存的管中。然后将腹膜液以250倍G离心5分钟，并储备上清液用于白细胞介素-6剂量。为了获得血清，将收集的血液样品以700倍G离心15分钟，并储备上清液用于淀粉酶和白细胞介素-6剂量。与假组的组织样本相比，诱导急性胰腺炎后12小时的胰腺组织分析显示受影响器官的强烈炎症。在对假和APTA组织样本进行苏木精曙红染色后的组织学检查表示了类似的结果。血清分析显示，与假组相比，AP组的血清淀粉酶和白细胞介素-6细胞因子浓度显着增加。同时，AP组腹膜腔液中白细胞介素-6浓度显著升高。该模型中胰腺炎的严重程度与输注的浓度、体积和压力以及急性胰腺炎诱导的时间成比例地取决于。在这种技术的帮助下，可以研究连续腹膜灌洗对严重急性胰腺炎的影响。

sodium-taurocholate-induced-severe-acute-pancreatitis-in-c57bl6-mice

Research

JoVE Journal

Medicine

3.7K Views.  University of São Paulo Medical School. This method can be used for reproducing the inflammatory events that occur in acute biliary pancreatitis. It is possible to study changes in pancreatic tissue in its early stages, which is generally not possible in the clinic. The main advantages of this technique are that it is quick and easy to reproduce for a trained researcher, and it offers similar outcomes to acute pancreatitis in humans.The method can be used to study pharmacological immune responses of drug molecules and other ...