Name: a mouse model for chronic pancreatitis via bile duct tnbs infusion
Uploaded: 2021-02-28T13:30:00
Description: Watch this Scientific Journal Video about A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion at JoVE.com

这项研究得到了退伍军人事务部（VA-ORD BLR&D功绩I01BX004536）的支持，国家卫生研究院向HW提供了1R01DK105183、DK120394和DK118529。我们感谢吴红菊博士分享技术经验。

所有程序均经南卡罗来纳医科大学动物护理和使用委员会和拉尔夫·约翰逊医疗中心批准。本研究使用了C57BL/6J雄性小鼠8-10周。老鼠被安置在一个标准的12光/12暗循环下，食物和水的自由。
1. 准备用于注射的 TNBS 解决方案
<ol><li>在 0.9% 盐中准备 10% 乙醇。将 10% 乙醇中的库存 TNBS（参见 材料表）溶解到 7.5 mM 的最终浓度，将 7.5 μL 的 TNBS 加入 1 mL 的 10% 乙醇中。 ...

<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=Histone+deacetylase+2+is+involved+in+&#181;-opioid+receptor+suppression+in+the+spinal+dorsal+horn+in+a+rat+model+of+chronic+pancreatitis+pain.">Histone deacetylase 2 is involved in &#181;-opioid receptor suppression in the spinal dorsal horn in a rat model of chronic pancreatitis pain.</a> Molecular Medicine Reports. 17 (2), 2803-2810 (2018).</li><li>Gui, 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=Trypsin+activity+governs+increased+susceptibility+to+pancreatitis+in+mice+expressing+human+PRSS1R122H.">Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.</a> The Journal of Clinical Investigation. 130 (1), 189-202 (2020).</li><li>Sun, Z., 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=Adipose+Stem+Cell+Therapy+Mitigates+Chronic+Pancreatitis+via+Differentiation+into+Acinar-like+Cells+in+Mice.">Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.</a> Molecular Therapy. 25 (11), 2490-2501 (2017).</li><li>Aghdassi, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+duct+pressure+in+chronic+pancreatitis.">Pancreatic duct pressure in chronic pancreatitis.</a> The American Journal of Surgery. 144 (3), 313-316 (1982).</li><li>Zhao, J. B., Liao, D. H., Nissen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pancreatitis:+can+it+be+translated+to+human+pain+study.">Animal models of pancreatitis: can it be translated to human pain study.</a> World Journal of Gastroenterology. 19 (42), 7222-7230 (2013).</li><li>Winston, J. H., He, Z. J., Shenoy, M., Xiao, S. Y., Pasricha, P. 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在小鼠中，TNBS输液诱发慢性胰腺炎的胆管输液在技术上具有挑战性，因为多达22.5%的小鼠在药物输液10天后3-4天内死亡。在这里，本报告在先前研究的基础上改进了程序，将早期小鼠死亡率降低到<10%。例如，药物量的增加（从 35 μL 增加到 50μL）可以确保药物到达整个胰腺。使用胰岛素注射器和较小的针头尺寸 （31G） 可减少胰腺导管的潜在损伤和胆汁渗入胆囊或腹部，这最有可...

慢性胰腺炎 （CP） 是一种慢性炎症性疾病，其特征是胰腺萎缩、纤维化、腹痛，以及最终失去外分泌和内分泌功能1.目前的医疗和外科治疗不是治疗性的，而是用来缓解疾病后果的症状：难治性腹痛、内分泌和外分泌功能障碍。因此，迫切需要更有效的治疗。动物模型提供了一个基本工具，以发展更好地了解这种疾病和研究潜在的治疗方法3。已开发了多个 CP 小鼠模型，其中通常使用纤维素和/或酒精模型。Cerulein，一种刺激胰腺分泌物的寡头肽，已被证明可重复诱导CP模型，其特征包括胰腺萎缩、纤维化等4种。另一种常见的模型使用L-精氨酸的连续注射，产生外分泌不足类似于在人类患者中观察到的5。CP也可以由完全或部分胰腺管粘连，以及胰腺导管高血压6，7诱导。尽管CP的动物模型种类繁多，但这些模型都没有有效地再现CP患者8经历的腹痛。
先前的研究表明，局部胰腺注射2，4，6-三硝基苯硫酸（TNBS）复制了CP患者9，10，11经历的持续疼痛。TNBS治疗的小鼠表现出腹部过敏和增加疼痛相关行为，以及"普遍的超敏"疼痛刺激，这种现象已经观察到在CP患者10。除了准确模仿CP疼痛，TNBS模型还复制了人类状况的其他病理特征，如纤维化，单核细胞渗透，以及用脂肪组织10，12替换细胞。然而，通过胆管输液是一个技术上具有挑战性的程序，在小鼠可能会导致死亡。据我们所知，没有视觉协议来显示胆管输液是如何进行的。在本文中，我们演示了 TNBS 的胆汁诱导输注产生 CP 鼠标模型的程序。这个程序将有助于产生有价值的动物模型，用于CP和其他胰腺疾病的研究，并可用于注入其他材料（例如，病毒，细胞）到胰腺13。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral buffered formalin v/v</td>
			<td>Fisher Scientific</td>
			<td>23426796</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads, sterile</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Anesthesia system</td>
			<td>VetEquip, Inc.</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride, injection</td>
			<td>Par Sterile Products, LLC</td>
			<td>NDC 42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>0553859A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute (200 proof), molecular biology grade</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine Micro Dissecting scissors 4&#8221; straight sharp</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps 4&#8221; extra delicate tip</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated pad</td>
			<td>Amazon</td>
			<td>B07HMKMBKM</td>
			<td></td>
		</tr>
		<tr>
			<td>Hegar-Baumgartner Needle Holder 5.25&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-7850</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe with 31-gauge needle</td>
			<td>BD</td>
			<td>324909</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine prep pads</td>
			<td>Fisher Scientific</td>
			<td>19-027048</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip applying forceps 5.5&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5410</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight strong curved 1x6mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5433</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight, 0.75mm clip width</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrylsulfonic acid solution, TNBS, 1M in H2O</td>
			<td>Millipore Sigma</td>
			<td>92822-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 4-0</td>
			<td>Med-Vet International</td>
			<td>MV-8683</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 5-0</td>
			<td>Med-Vet International</td>
			<td>MV-8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, 0.9% intravenous solution</td>
			<td>VWR</td>
			<td>2B1322Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape, sterile</td>
			<td>Med-Vet International</td>
			<td>DR1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassette</td>
			<td>Fisher Scientific</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Frey filaments</td>
			<td>Bioseb</td>
			<td>EB2-VFF</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model for chronic pancreatitis via bile duct tnbs infusion

慢性胰腺炎 （CP） 是一种复杂的疾病，涉及胰腺炎症和纤维化、腺萎缩、腹痛和其他症状。已开发了几种啮齿动物模型来研究CP，其中胆管2，4，6-三硝基苯硫酸（TNBS）输液模型复制了CP中神经病变疼痛的特征。然而，胆管药物输注在小鼠是技术上具有挑战性的。此协议演示了胆管 TNBS 输注过程，用于生成 CP 鼠标模型。TNBS通过十二指肠的瓦特安普拉注入胰腺。该协议优化了药物体积、外科手术技术和药物处理过程。TNBS 治疗的小鼠表现出 CP 的特征，如体重和胰腺体重减轻、疼痛相关行为的变化和胰腺形态异常所反映。有了这些改进，与TNBS注射相关的死亡率是最小的。此过程不仅在生成胰腺疾病模型方面至关重要，而且对当地胰腺药物的提供也很有用。

胆管输液程序进行了优化，以降低与此程序相关的小鼠死亡率。TNBS 首次提供总容量为 35 μL 或 50 μL。在50微升的体积内注射TNBS可以到达整个胰腺，并诱发更均匀的疾病表型（图1B）。此外，使用31G针头注射胰岛素注射器的TNBS可以更好地控制输液速度相对于常规注射器和针头的大小。与准备时间超过一小时的TNBS相比，储存在冰上并在药物制剂后一小时内使?...

Watch this Scientific Journal Video about A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion at JoVE.com

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

慢性胰腺炎 （CP） 是一种以胰腺炎症和纤维化为特征的疾病，通常与难以克服的腹痛有关。本文侧重于改进技术，通过胆管输液产生CP的小鼠模型与2，4，6-三硝基苯硫酸（TNBS）。

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

慢性胰腺炎是一种复杂的疾病，涉及胰腺炎症、纤维化、腹痛和其他症状。胆管TNBS输液模型复制慢性胰腺炎患者观察到的神经病变疼痛。这种胆管TNBS输液方案经过优化，在小鼠中诱导慢性胰腺炎模型的产生。由于小鼠的胆管药物融合在技术上具有挑战性，其可视化将有助于研究人员对该方法的新发现。手术前两周和手术后三周，使用冯弗雷单体施加不同程度的力，以提升顺序对上腹部区域的8至10周大，C57黑6小鼠，10次，每次应用1至2秒。考虑提高腹部的缩回或舔作为积极的反应。接下来，装载装有 31 量针的胰岛素注射器，该注射器配有 50 微升新制备的 0.75% TNBS 溶液。将第二个注射器与 50 微升 10% 乙醇一起装在盐水中作为车辆控制，并将两个注射器放在冰上，防止光线照射。在确认对踏板反射缺乏反应后，通过剃须或涂抹脱毛霜将麻醉小鼠的腹部区域的头发取出，并将鼠标放在解剖显微镜下的加热手术垫上。将兽药膏涂在每只眼睛上，用三个连续的2%碘和3个70%的乙醇湿巾对手术部位进行消毒，使用微型剪刀连续运动0.5至1厘米，并用棉签轻轻地暴露十二指肠，定位普通胆管。为了防止治疗流入肝脏和胆囊，使用显微镜在近界普通导管上放置一个直的微血球，并通过桶的将 TNBS 装载的注射器的针头插入胰腺导管。使用弯曲的微血球固定针头，并在一分钟内将溶液注入胰腺导管。当所有溶液都交付时，小心地取下微夹，并确认十二指肠已恢复到原来的位置。在腹腔关闭之前，加入500微升36至37摄氏度的无菌盐水，以帮助十二指肠恢复其原来的位置，并协助恢复永久性。使用连续缝合与五O缝合，以关闭肌肉层的切口，并关闭皮肤使用中断的缝合与四O缝合。然后将鼠标放在加热垫上的回收笼中，进行监控，直到完全恢复。在手术后的第一周，每天监测小鼠的健康和行为，以发现不适的迹象，如发声、驼背姿势或减少运动，每隔一天测量一次体重。观察到，注射50微升墨水，证明结果导致整个胰腺的渗透，允许诱导一个更同质的疾病表型。在这个具有代表性的分析中，在手术后的头三天，对照组的小鼠在逐渐恢复之前减掉了原体重的6%左右，到第21天体重增加了100%以上。相比之下，接受TNBS治疗的老鼠在手术后的头五天里减掉了原体重的15%，到第21天体重恢复到原来的100%以下。与对照小鼠相比，TNBS注射的动物在手术后的第二周和第三周表现出腹部机械过敏性增加。与从对照动物身上采集的胰腺相比，TNBS小鼠的胰腺体型和体重显著减少，这表明TNBS治疗的动物的胰腺萎缩明显，与患有严重和长期慢性胰腺炎的人类发现一致。对控制乙醇治疗小鼠胰腺组织部分的组织学分析显示，其形态正常，而TNBS治疗小鼠的组织部分则表现出空隙实现，大量丧失了被脂肪细胞渗透和纤维化所取代的细胞。防止注射的 TNBS 进入膀胱和肠道很重要，因为 TNBS 具有组织破坏性影响。输液期间，保持手尽可能稳定，以避免胆管穿刺作用。除了诱导慢性胰腺炎的发展，这种胆管输液小鼠艺术可用于其他胰腺疾病模型的发展。这种TNBS慢性胰腺炎小鼠模型可用于研究慢性胰腺炎的发病机制和治疗以及其他胰腺疾病。

Research

JoVE Journal

Medicine

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

沃顿商学院的导管插管通过小鼠颌下腺唾液腺

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

科研

医学

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

树脂浇铸和3维成像的小鼠门静脉或肝内胆管系统

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

A Model of Chronic Nutrient Infusion in the Rat

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

胆管结扎小鼠：感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

胰腺管输液：药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

回肠致胆汁积滞在小鼠肠道中

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...