Name: dissection of the mouse pancreas for histological analysis and metabolic profiling
Uploaded: 2017-08-19T14:00:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling at JoVE.com

麦承认支持这项工作从国立卫生研究院和国家癌症研究所资助编号-1R15CA152985 01A1。这个项目也得到了奖学金计划和迈阿密大学学者暑期的迈阿密大学博士学位本科机会迈阿密大学本科教学科研奖励计划。

在视频中进行，如下所述的程序已获机构动物护理和使用委员会 (IACUC) 在迈阿密大学。
 1.制备及刺激试验 <ol><li>建立两个不同的区域，为外科手术、 手术台和术后表。舞台这两个领域与所有材料和必要的用具。
	<ol><li>阶段的操作表排气罩下。把桌子布置与设备允许程序的连续和不受阻碍地性能的方式。</li>
		<li>建立一个术后的表在同一个房间里和附近的主表的操作。保持这两个表作为整个过程的无菌环境。</li>
	</ol></li> <li>以下的地方用品在手术台上: 一个玻璃罐盖、 一 15 毫升管、 手术剪刀、 70%乙醇、 两个泡沫板、 两个钳、 两个 1 毫升 21 计注射器，两个 50 毫升管、 一个离心管一挤压瓶一对一个低温瓶、 四个手术垫、 保龄球、 一个配药的消毒湿巾，尖锐物品容器。</li>
	<li>在术后表上放置以下物品: 一个分析天平，一个杜瓦 L 月 4 日的液态氮，浅全口杜瓦瓶、 浮动的微细管架、 一把手术剪刀、 两个钳、 四个低温瓶和一个配药的无菌湿巾。</li>
	<li>一个 50 毫升和 75%卷一 15 毫升管装满福尔马林。</li>
	<li>在每个角落里，用一根针贴上一个手术垫到近似 30 厘米 × 30 厘米泡沫板作为夹层板。在操作过程中使用的剩余的四个针脚。准备将器官转移到术后表手术垫小泡沫板。 
	警告: 异氟烷 （99.9%) 是一种有毒的化学，和应使用在排气罩以确保最高安全级别的从清除废物的麻醉气体 8。有关研究人员开放 drop 方法用异氟醚的使用相关的风险的其他信息可以发现在一篇文章由泰勒和穆 8.</li>
	<li>将一个手术垫放入玻璃罐，用几滴异氟醚 （99.9%) 浸泡和上方以防止直接接触的异氟醚与鼠标放置一张纸巾。同样，使用手术垫线剩余管和浸泡用几滴异氟醚和附加垫置于顶端，以防止直接接触鼠标和异氟醚。</li>
	<li>倒进浅的宽嘴里杜瓦瓶直到最高填充线达到液态氮。</li>
	<li>为剥离至麻醉选择鼠标的地方分庭，即 玻璃罐用垫浸透几滴异氟烷 （99.9%) 盖上盖子，~ 1 分钟 
	注意: 此时间而有所不同从老鼠。一旦鼠标是无意识的删除它从分庭，然后将其放置在操作盘。</li>
	<li>东方鼠标，它躺在腹侧和与它的头朝外的科学家。将内管内衬与异氟醚 （99.9%)，滴几滴手术垫头部放和执行刺激测试脚夹以确保鼠标是对刺激反应迟钝。
	<ol><li>如果这个测试失败，并且鼠标响应脚夹点测试，请重复步骤 1.8。</li>
	</ol></li></ol> 2。最初的切口、 心脏穿刺，和安乐死 <ol><li>引脚鼠标到外科泡沫板的四肢和湿用 70%乙醇鼠标腹侧。</li>
	<li>用钳夹附近的尿道口的毛皮和轻微向上拉。用通过腹腔从尿道开放，中线开始和结束在下巴的手术剪刀做一个切口。
	<ol><li>在起始点附近的初始切口，抢一侧的毛皮的镊子和使另一个切口用剪刀沿对角线方向向下和向背爪子。</li>
		<li>重复此步骤以同样的方式在另一侧. 
		注意: 毛皮可以牵制打造一个更广泛的开头，但不是必要。</li>
	</ol></li> <li>找到心并清除心包，是周围的心，以避免堵塞针头的囊。
	<ol><li>钳子抓住心包膜，然后用剪刀剪。通过仔细地将针头插入心脏跳动执行心脏穿刺法，慢慢开始收回柱塞。</li>
		<li>最佳采血，使用针的柱塞模仿心脏的泵血动作，避免太快绘图。 
		注: 通常约 1 毫升的血可以收集。</li>
		<li>完成后采血，消除血液离心管和注射器的锐器盒内处置。</li>
		<li>后心脏穿刺口，实施安乐死删除附件连接心脏。 
		注: 肝素抗凝血剂，未添加到在此过程中心脏穿刺法，可以使血液凝结在这特定的研究中，血清集合之前注射器。但是，如果研究者想要防止血液凝固，血浆收集，肝素可以添加到前心穿刺注射器。</li>
	</ol></li> <li>如果这项研究涉及基因分型的鼠标，用剪刀剪耳朵的一部分并将放入离心管的基因型核查。</li></ol>
 3.胰腺提取 <ol><li>定位鼠标左侧的胃。轻轻地 （以避免撕裂） 开始使用两个钳，从胃部和十二指肠分隔胰腺。 
	注: 分离胰腺从胃和肠道时, 它是非常重要，轻轻地用镊子指导器官隔离的胰腺组织，不粉碎或撕裂胰腺的镊子。
	<ol><li>继续分离胰腺从小肠空肠和回肠部分，最后由大肠盲肠。</li>
	</ol></li> <li>在盲肠，复位钳，继续沿着剩余结肠直肠走向胰腺分离。 
	注: 在这一点上，它是方便剪切和删除的部分从胃里到该地区紧接直肠结肠。</li>
	<li>定位胰腺和附加脾。滑向右边的鼠标胰腺。用钳要完全分离胰腺和附加广义的脾脏分隔的胰腺和胸腔内的剩余联系。</li>
	<li>删除胰腺和展开检查。离开附加到用于识别目的胰腺脾。
	<ol><li>删除所有结缔组织、 脂肪和肠系膜组织从胰腺。 
		注: 这种组织是白的颜色，因此可以很容易区分，是粉色的颜色的胰腺组织。这是特别重要，如果整个胰腺需要被删除。例如，如果胰腺需要加以权衡和比较体重或动物群体之间。在 Ptf1a cre / +;LSL Kras G12D / + 小鼠模型，特别是在较旧的月份，硬纤维胰腺组织可能存在。在这种情况下，必须进行仔细去除的胰腺为肠道可能在肿瘤组织中交错式。在先进的情况下，异常脾和肝组织可能也会出现。</li>
	</ol></li> <li>如果需要，在这一点上删除其他的器官。</li></ol>
 4.数据收集和存储 <ol><li>解压后的器官，将样品移至术后区域保存。</li>
	<li>权衡每个器官和将它们放置到他们各自的低温瓶。 
	注: 沿机智h 质量的每个示例中，任何违规行为应记录以备将来参考。</li>
	<li>一旦重质器官放入液氮为管理单元冻结。</li>
	<li>后冻结的管理单元，存储长期储存在-80 ° C 器官。</li>
	<li>地方福尔马林保存样品在台阶顶上一夜之间，和第二天早上从福尔马林更改他们的解决方案，为 70%乙醇。 
	注意: 这些样品应储存在 4 ° C 的长期存储。</li>
	<li>为长时间存放，冻结在-80 ° C.血液和耳朵冲床。血清收集从血液，使血液凝结为 30 分钟，然后离心它。删除使用吸管的血清部分，然后存储在-80 ° C.</li>
</ol> 5。清理一下 <ol><li>Sanitize 所有解剖工具与消毒湿巾。帽管内衬异氟醚浸泡手术垫。用新鲜的手术垫替换手术垫泡沫板上。处置的部分鼠标不收集设施每 &#39; s 动物处置政策。</li>
</ol>

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作者没有透露。

重要的意义，现有的方法 虽然其他非正式的视频鼠标夹层动脉瘤存在，视频本文提供专业质量第一、 同行评审、 直观示范的所有详细的步骤所需的提取和收获的小鼠胰腺的解剖10.正在主要器官代谢活性和胰岛素的胰腺生产、 夹层和收获的胰腺允许保存的生理特性11。通过隔离胰腺，可能对样品进行未来的分析。这个程序允许从其他组织在相同?...

鼠标已成为人类胰腺癌1，2重要动物模型。在 Ptf1acre / +;LSL KrasG12D / +小鼠模型，大鼠肉瘤 (K-Ras) 癌基因激活只在胰腺，导致胰腺，胰腺上皮内肿瘤 (PanINs)，这一进展被称为癌前病变引发的克里斯汀对胰腺导管腺癌，通常所说的 PDACs3。这个鼠标模型系统提供了一个人类胰腺癌4，5，与 PanINs 出现在首五个月的生活和频繁地进展 PDAC 内的附加优势的最佳动物模型单 44，5，而胰腺癌最频繁地出现在人类身上 60-70 岁的年龄。
由 Ptf1a 淋巴结清扫术胰腺提取cre / +;LSL KrasG12D / +小鼠不同年龄允许详细纵向组织学检查癌症发展的胰腺，从最早的 PanIN 阶段通过进展到 PDAC3，4,5.在年龄从五到十五个月收获胰腺可也用于准备组织提取物表征在胰腺4代谢在从健康到病变组织的过渡过程中发生的全球变化6，7。
本文介绍了一个完整的视觉指南执行鼠标胰腺提取所需的步骤并提供指导方针的胰腺，进一步分析存储。本指南将同样宝贵，为个人进行研究其他胰腺的疾病，包括 I 型糖尿病，和应该是特别有用的学生和研究人员的新研究涉及收获的小鼠胰腺使用清扫术代谢轮廓分析或组织学分析。

<table><tbody><tr><td>Glass Jar&amp;nbsp;</td><td>Corning</td><td>3140-150</td><td>The glass jar used in the video has been discontinued.&amp;nbsp; This is its replacement.</td></tr><tr><td>Lid of Glass Jar</td><td>Corning</td><td>9985-150</td><td>The glass jar lid used in the video has been discontinued.&amp;nbsp; This is its replacement.</td></tr><tr><td>15 mL Falcon Tubes</td><td>Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fisher Scientific</td><td>9201</td><td></td></tr><tr><td>Squeeze bottle&amp;nbsp;</td><td>Fisher Scientific</td><td>03-409-10DD</td><td></td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>22-032-103</td><td></td></tr><tr><td>Formalin</td><td>Fisher Scientific</td><td>245-684</td><td></td></tr><tr><td>Foam Boards</td><td>Therapak</td><td>562908</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>200205SHN</td><td></td></tr><tr><td>1 mL 21G Syringes</td><td>BD Biosciences</td><td>309624</td><td></td></tr><tr><td>50 mL Falcon Tubes</td><td>Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>2.0 mL Microcentrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>02-681-258</td><td></td></tr><tr><td>Surgical Pads</td><td>Fisher Scientific</td><td>S67011</td><td></td></tr><tr><td>T-Pins Length:&amp;nbsp; 2&quot;&amp;nbsp;</td><td>Advance Store Products</td><td>X32T-05</td><td></td></tr><tr><td>Sterilizing Wipes</td><td>Professional Disposables International Inc.</td><td>Q85084</td><td></td></tr><tr><td>Sharps Container</td><td>Fisher Scientific</td><td>14-827-122</td><td></td></tr><tr><td>Analytical Balance</td><td>Marshall Scientific</td><td>ME-AE200</td><td></td></tr><tr><td>4L Dewar</td><td>Taylor-Wharton</td><td>4LD</td><td></td></tr><tr><td>Shallow Wide Mouth Dewar</td><td>Fisher Scientific</td><td>F3087-V</td><td></td></tr><tr><td>Floating Microtube Rack</td><td>VWR</td><td>60986-100</td><td></td></tr><tr><td>Cryogenic Vial 1.2 mL, Sterile</td><td>Fisher Scientific</td><td>10-500-25</td><td></td></tr><tr><td>Isothesia (Isoflurane)&amp;nbsp;</td><td>Henry Schein Animal Health</td><td>050033</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Wright Brothers</td><td>NIT-60-XX</td><td></td></tr><tr><td>Mouse Kras Strain</td><td>The Jackson Laboratory</td><td>OO8179</td><td></td></tr><tr><td>Mouse Cre Strain</td><td>MMRRC</td><td>OOO435-UNC</td><td></td></tr></tbody></table>

dissection of the mouse pancreas for histological analysis and metabolic profiling

我们一直在调查胰腺特异性转录因子、 1a-cre 重组酶;脂氧合酶-停止-脂氧合酶-克里斯汀大鼠肉瘤、 甘氨酸对天冬氨酸在 12 密码子 (Ptf1acre / +;LSL-KrasG12D / +) 鼠标应变作为人类胰腺癌模型。我们当前的研究的目标是找出新型代谢生物标记物的胰腺肿瘤。我们进行代谢过程的尿液、 粪便、 血液，和胰腺组织提取物，以及胰腺期癌的组织学分析。小鼠胰腺不是定义良好的实质器官，像人类，而是弥漫性分布的软组织不容易标识由个人熟悉鼠标内部解剖或有很少或没有表演经验的个人鼠标器官解剖。这篇文章的目的是提供一个详细的逐级直观示范指导新手在小鼠胰腺切除的解剖。这篇文章应该向学生和研究人员新需要收获的小鼠胰腺剥离术治疗代谢轮廓分析或组织学分析的研究尤为重要。

图 1显示的操作环境区的概述和图 2显示了岗位操作区域。虽然此设置提供最小数量的设备和分期，个人可以选择改变了这一点，最适合个人需要。议定书 》 应根据实验的具体需要进行优化。此过程以终止的一只老鼠，需要适当的狗狗会被安乐死9生活的方式进行。当研究者是准备好了时，鼠标放入麻醉室与异...

Watch this Scientific Journal Video about Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling at JoVE.com

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

本文视频提供了详细的演示了从一只老鼠成功删除胰腺解剖组织学分析和代谢过程所需的程序。

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

dissection-mouse-pancreas-for-histological-analysis-metabolic

Research

JoVE Journal

Medicine

36.5K 次观看.  Miami University. 本文视频提供了详细的演示了从一只老鼠成功删除胰腺解剖组织学分析和代谢过程所需的程序。

视频: Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

三维细胞培养中胰腺肌囊内增生的模拟与操纵

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

科研

癌症研究

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

发育生物学

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

胰腺组织解剖以分离活的单细胞

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

在小鼠胰腺β-细胞团的原位定量

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

生物学

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

医学

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

免疫与感染

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (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. ...

Acinar-to-Ductal Metaplasia Induction: A Method to Study Acinar Cell to Ductal Cell Differentiation in 3D Ex Vivo Culture

Source: Fleming Martinez, A. K. et. al. <a href="https://www.jove.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
This video describes the technique of differentiating pancreatic acinar cells to ductal cells in ex vivo 3D culture. This helps to understand the mechanisms regulating the acinar-to-ductal metaplasia (ADM), a key process in the early development of pancreatic cancer.

腺泡到导管化生诱导:一种在 3D 离体培养中研究腺泡细胞到导管细胞分化的方法

Source: Fleming Martinez, A. K. et. al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...