Name: laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo
Uploaded: 2018-03-08T13:00:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

这项工作得到了北京协和医学院青年基金和中央大学基础研究基金的资助 (3332015200 号赠款)。

所有动物实验都是按照所有相关的指导方针、法规和监管机构执行的。目前正在示范的这项议定书是在北京协和医学院 (PUMC) 的微循环动物伦理委员会 (IMAEC) 的指导和批准下进行的。
1. 动物
<ol><li>实验开始前, 在 5%-小时的光暗循环下, 保持每笼三 BALB 小鼠, 控制温度 (24 1 摄氏度) 和湿度 (55 @ 12)。允许老鼠免费获得正常的食物和水。</li>
	<li>将小鼠随机分为非糖尿病对照组和糖尿病组。用每只老鼠的身体质量准确地称量每只老鼠并计算注射量。</li>
	<li>在4小时内快速的小鼠在链脲佐菌素 (STZ) 注射和提供常规水正常的实验日1。</li>
	<li>在 pH 值4.3 上制备0.1 米柠檬酸钠缓冲液。将1毫升的溶液放入一个1.5 毫升的离心管中, 用铝箔包裹离心管, 以避免光线暴露。</li>
	<li>在使用前, 将在柠檬酸钠缓冲液 (pH 4.3) 中的 STZ 分解为5毫克/毫升的最终工作浓度。</li>
	<li>用1毫升注射器和25克针, 用40毫克/千克的剂量, 给糖尿病组腹腔注射 STZ 的老鼠。用相同体积的柠檬酸钠缓冲液注射非糖尿病控制的小鼠 (pH 为 4.3)。</li>
	<li>把老鼠放回笼子里, 用普通的食物和10% 的蔗糖水供给它们。</li>
	<li>在实验日2到 5 (即,连续4天) 重复步骤 1.3-1.7。</li>
	<li>在最后一次注射后, 用常规水代替10% 的蔗糖水。</li>
	<li>将小鼠快速6小时, 但给他们免费获得水, 并测量他们的血糖水平九天后 (实验日 14)。从尾部静脉收集血液样本, 用血糖监测系统确认高血糖。 注: 血糖水平 > 200 毫克/dL 的老鼠被认为是糖尿病。</li>
</ol>2. 文书的编写
<ol><li>用软的非研磨布清洁激光多普勒装置探头尖端和探头接头的光学表面, 以除去任何灰尘或微粒。将电缆插入仪器的端口 (图 1A)。</li>
	<li>通过允许焊剂标准与实验环境 (室温, 通常为30分钟) 的热平衡来装配校准架。轻轻摇动焊剂标准十年代, 让它休息2分钟。</li>
	<li>将通量标准容器放置在校准基座的中间。将夹具调整到最大高度, 并将夹具中的探头固定, 使其向下指向容器。确保磁通标准正确定位在探头下方。</li>
	<li>慢慢地将探头向下移动, 直到刀尖正确地浸入通量标准中。选择并按下激光多普勒装置的 "校准", 选择探头连接到的工作通道。运行校准程序, 直到 "校准成功" 的通知显示在屏幕上的激光多普勒仪器。</li>
	<li>使用探头持有者来保护探头。手动保护探头以避免移动。</li>
	<li>保持实验室恒温 (24 1 摄氏度) 和湿度 (~ 50-60%)。</li>
	<li>在进行实验前, 关闭任何外部光源 (如荧光灯和光斑灯), 以避免外部光线诱发的变化。</li>
</ol>3. 动物的准备
<ol><li>对手术器械进行高压灭菌, 在使用前使其降温至室温。</li>
	<li>用激光多普勒检测胰岛微血管血管舒缩, 给小鼠10分钟的适应实验环境。</li>
	<li>用1毫升3% 戊巴比妥钠填充1毫升注射器。注射戊巴比妥钠溶液 (75 毫克/千克 ip) 麻醉小鼠。</li>
	<li>用预湿的医用纱布遮住老鼠的眼睛, 以防干燥。</li>
	<li>确保鼠标完全失去知觉, 不再响应尾巴或 hindfoot 捏钳。在麻醉和手术中每15分钟监测麻醉情况, 必要时补充10% 的戊巴比妥溶液的初始注射量。</li>
	<li>在动物下方放置一个半绝缘层的加热垫, 将动物置于仰卧位置, 将其转移到激光多普勒装置的工作台。用手术胶带将鼠标固定在工作平台上。</li>
	<li>用 betadine 拭去老鼠的腹部皮肤, 然后用75% 乙醇擦拭腹部清洁。</li>
	<li>注射2% 利多卡因/0.5% 布比卡因 (50/50) 混合物皮下。 在纱布海绵的中心切一个直径为3厘米的孔。用纱布海绵覆盖腹部区域。</li>
	<li>用手术刀或皮剪刀在腹部中线上用镊子抬起腹部皮肤。</li>
	<li>用钳和切口抓住底层肌肉进入腹腔。不要伤害任何器官。将皮肤和底层肌肉折叠在胸前, 露出腹腔。用一双钝鼻钳轻轻地将胰体和脾脏暴露出来。</li>
</ol>4. 数据获取分析
<ol><li>通过点击 "文件" → "新建" 来创建一个新的测量文件, 运行激光多普勒装置的软件。要配置已连接的监视器, 请在 "常规" 选项卡下, 将监视持续时间设置为 "自由运行"。使用出厂默认值 "监视器" 选项卡. 单击 "下一步"。</li>
	<li>在 "显示设置" 对话框中设置图形显示。通过检查各个框, 选择 "通量、轻质、速度" 通道。选择以下参数: "通道的数据源" 和 "标签、单位和颜色"。单击 "下一步"。</li>
	<li>在 "文件信息" 对话框中输入有关主题和度量 (即名称和主题编号、运算符、监视时间、注释、等) 的用户信息, 然后单击 "下一步" 以完成测量配置。 注意: 测量窗口由软件自动创建 (图 1B)。</li>
	<li>手动将电极推进胰腺。确保探头与胰腺组织之间的距离在1毫米以内。不适当的距离会人为地增加或减少血流读数。</li>
	<li>单击 "开始" 工具栏图标开始记录微血管血流灌注单元 (PU) 数据。每次运行时连续收集 PU 数据1分钟。单击 "停止" 以停止测量。选择 "文件" → "另存为" 以命名并保存完成的测量文件。</li>
	<li>每次运行后手动重新定位探头, 以避免附加效果和收缩和松弛容量的局部耗尽。重复步骤 4.1-4.4 收获多点 (即,三随机选择点从胰腺组织) 微血管 PU 数据为每只老鼠。测量非反射板的 PU 数据作为基线控制。</li>
	<li>用缝合闭合腹部肌肉层和皮肤层。实验后把动物放在干净的笼子里。</li>
	<li>把恢复笼半放在加热垫上, 保持动物的保暖。 注意: 注意保暖、卫生、液体和食物摄取, 以及感染。对2毫克/千克卡洛芬的小鼠进行48小时的术后疼痛管理。 当观察到小鼠处于严重的疼痛或窘迫状态时, 注射150毫克/千克戊巴比妥钠, 以此来进行安乐死。</li>
</ol>5. 微血管血管舒缩参数的计算
<ol><li>使用激光多普勒软件的 "导出" 命令将时间和 PU 原始数据导出为 xlsx 文件, 并在电子表格中打开该文件。</li>
	<li>计算平均基线灌注单元 (PUb) (请参见步骤 4.6)。</li>
	<li>计算平均血液灌注 (pua) 的测量值为1分钟, 如下所示: 平均血液灌注 (pua) = 浦-浦b (等式 1)。</li>
	<li>计算每1分钟测量的频率 (周期/分钟)。 注: 微血管血管舒缩的频率定义为每分钟微血管血管舒缩波发生的峰值数。</li>
	<li>计算每1分钟测量的振幅 (ΔPU)。<ol>
<li>计算微血管血管舒缩的振幅为最大值 (浦max) 和最小值 (浦min) 的差异: 振幅 (ΔPU) =PU最大-浦最小 (等式 2)。</li>
	</ol>
</li>
	<li>计算每1分钟测量的相对速度 (PU)。</li>
</ol>

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作者没有什么可透露的。

在涉及微血管功能障碍的病例中 (例如,糖尿病、急性胰腺炎、周围微血管疾病、等), 一些疾病导致血流减少。除了血液流动的变化, 还有重要的指标, 如微血管血管舒缩, 反映微循环的功能状态。具体指标, 微血管血管舒缩, 一般被定义为微血管的音调在微血管床上的振荡。在本协议中, 微血管血流灌注监测系统允许对微血管血管舒缩的功能状态进行直接的可视化和定量分析。我们的微?...

微血管血管舒缩作为微循环功能状态的一个参数, 负责氧气、营养素和荷尔蒙的传递和交换, 对去除代谢产物, 如二氧化碳和细胞浪费至关重要。1. 微血管血管舒缩还调节血液流量分布和组织灌注, 从而影响局部微循环血压和炎症反应, 可诱发多种疾病的水肿。因此, 微血管血管舒缩对维持器官的生理功能非常重要2, 3, 4, 组织和组件单元格.微血管血管舒缩的损害可能是发展微循环相关疾病的关键步骤之一5。
激光多普勒初步发展为观察和定量在微循环研究领域的6。该技术与其他技术方法 (例如,激光散斑7, 经皮氧,等) 一起被认为是评价微循环血流的黄金标准。根据多普勒移位原理, 用激光多普勒装置测定局部微循环 (即,毛细血管, 动脉, 静脉,等) 的血流灌注的基本原理。当光粒子遇到微血管中的运动血细胞时, 受激发射光的波长和频率会发生变化, 或者保持不变。因此, 在微循环中, 血细胞的数量和速度是与多普勒转移光的大小和频率分布有关的关键因素, 而微血管血流方向则无关紧要。使用不同的方法, 各种组织已被用于微循环研究, 包括 mesenteries 和背皮脂室的小鼠, 大鼠, 仓鼠, 甚至人类8。然而, 在本协议中, 我们重点研究胰岛微血管血管舒缩的功能状态, 用激光多普勒和自制的评价参数系统进行评价。
胰岛微循环主要由胰岛微血管构成, 表现出鲜明的特征。胰岛毛细血管网络显示五倍以上的密度比毛细管网络的外分泌对应9。胰岛内皮细胞提供输入葡萄糖和传播胰岛素的导管, 将氧气传递给胰岛β细胞代谢活性细胞。此外, 新的证据也表明, 胰岛微血管不仅涉及调节胰岛素基因表达和β细胞生存, 而且还影响β细胞功能;促进β细胞增殖;并产生一些血管活性、血管生成物质和生长因子10。因此, 在这方面, 我们推断胰岛微血管血管舒缩的功能状态可能影响胰岛β细胞功能, 并参与疾病的发病机制, 如急性/慢性胰腺炎, 糖尿病等与胰腺有关的疾病。
分析胰岛微血管血管舒缩功能状态的病理变化可能是确定胰岛微循环对上述疾病贡献的可行策略。详细的分步过程描述的方法, 以确定胰岛微血管血管舒缩在体内提供这里。然后, 将在代表性结果中显示典型的度量值。最后, 在讨论中突出显示方法的优点和限制, 以及进一步的应用程序。

<table><tbody><tr><td>MoorVMS-LDF2</td><td>Moor Instruments</td><td>GI80</td><td>PeriFlux 5000 (Perimed Inc.) can be used as an alternative apparatus to harvest data</td></tr><tr><td>MoorVMS-PC Software</td><td>Moor Instruments</td><td>GI80-1</td><td>Software of MoorVMS-LDF2</td></tr><tr><td>Calibration stand</td><td>Moor Instruments</td><td>GI-cal</td><td>Calibration tool</td></tr><tr><td>Calibration base</td><td>Moor Instruments</td><td>GI-cal</td><td>Calibration tool</td></tr><tr><td>Calibration flux standard</td><td>Moor Instruments</td><td>GI-cal</td><td>Calibration tool</td></tr><tr><td>One Touch UltraEasy glucometer</td><td>Johnson and Johnson</td><td>#1955685</td><td>Confirm hyperglycemia</td></tr><tr><td>One Touch UltraEasy strips</td><td>Johnson and Johnson</td><td>#1297006</td><td>Confirm hyperglycemia</td></tr><tr><td>Streptozotocin</td><td>Sigma-Aldrich</td><td>S0130</td><td>Dissolve in sodium citrate buffer (pH 4.3)</td></tr><tr><td>Pentobarbital sodium</td><td>Sigma-Aldrich</td><td>P3761</td><td>Working concentration 3 %</td></tr><tr><td>Ethanol</td><td>Sinopharm Inc.</td><td>200121</td><td>Working concentration 75 %</td></tr><tr><td>Sucrose</td><td>Amresco</td><td>335</td><td>Working concentration 10 %</td></tr><tr><td>Medical gauze</td><td>China Health Materials Co.</td><td>S-7112</td><td>Surgical</td></tr><tr><td>Blunt-nose forceps</td><td>Shang Hai Surgical Instruments Inc.</td><td>N-551</td><td>Surgical</td></tr><tr><td>Surgical tapes</td><td>3M Company</td><td>3664CU</td><td>Surgical</td></tr><tr><td>Gauze sponge</td><td>Fu Kang Sen Medical Device CO.</td><td>BB5447</td><td>Surgical</td></tr><tr><td>Scalpel</td><td>Yu Lin Surgical Instruments Inc.</td><td>175C</td><td>Surgical</td></tr><tr><td>Skin scissor</td><td>Carent</td><td>255-17</td><td>Surgical</td></tr><tr><td>Suture</td><td>Ning Bo Surgical Instruments Inc.</td><td>3325-77</td><td>Surgical</td></tr><tr><td>Syringe and 25-G needle</td><td>MISAWA Inc.</td><td>3731-2011</td><td>Scale: 1 ml</td></tr></tbody></table>

laser doppler: a tool for measuring pancreatic islet microvascular vasomotion in vivo

微血管血管舒缩作为微循环的功能性状态, 对于氧气和养分的输送以及二氧化碳和废弃物的去除具有重要意义。微血管血管舒缩的损伤可能是微循环相关疾病发展的关键步骤。此外, 高血管化胰岛适应内分泌功能。在这方面, 似乎可以推断胰岛微血管血管舒缩的功能状态可能影响胰岛功能。胰岛微血管血管舒缩功能状态的病理变化分析可能是确定胰岛微循环对相关疾病如糖尿病的贡献的可行策略,胰腺炎,等。因此, 本协议描述使用激光多普勒血流监测仪确定胰岛微血管血管舒缩的功能状态, 建立参数 (包括平均血流灌注、振幅、频率和相对胰岛微血管血管舒缩的速度, 用于评价微循环功能状态。在链脲佐菌素诱导的糖尿病小鼠模型中, 我们观察到胰岛微血管血管舒缩功能状态受损。总之, 这种评估胰岛微血管血管舒缩在体内的方法可能揭示与胰岛疾病有关的机制。

在图 1A中显示了配备半导体激光二极管的微血管血管舒缩测量激光多普勒装置的照片。用户界面软件显示在图 1B中。采用上述方法, 对非糖尿病对照和糖尿病小鼠胰岛微血管血管舒缩的血流动力学参数进行检测。许多技术, 包括激光多普勒血流仪, 反射和散射光, 红外光谱和成像技术, 已被用来研究微血管血管舒缩, 因为它是首次?...

Watch this Scientific Journal Video about Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo at JoVE.com

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

胰岛微血管血管舒缩调节胰岛血液分布, 维持胰岛β细胞的生理功能。本协议描述了使用激光多普勒显示器测定胰岛微血管血管舒缩在体内的功能状态, 并评估胰岛微循环对胰腺相关疾病的贡献。

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

laser-doppler-tool-for-measuring-pancreatic-islet-microvascular

Research

JoVE Journal

Medicine

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

8.2K 次观看.  Chinese Academy of Medical Sciences & Peking Union Medical College. 胰岛微血管血管舒缩调节胰岛血液分布, 维持胰岛β细胞的生理功能。本协议描述了使用激光多普勒显示器测定胰岛微血管血管舒缩在体内的功能状态, 并评估胰岛微循环对胰腺相关疾病的贡献。

视频: Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

科研

医学

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, ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

生物学

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

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

激光多普勒灌注成像在鼠标欣德利姆

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

研究人类微血管功能障碍的新机制

Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally invasive technique used to investigate mechanisms of vascular smooth muscle and endothelial function in the cutaneous circulation. This technique allows for the pharmacological dissection of the pathophysiology of microvascular endothelial dysfunction as indexed by decreased nitric oxide-mediated vasodilation, an indicator of cardiovascular disease development risk. In this technique, a microdialysis probe is placed in the dermal layer of the skin, and a local heating unit with a laser Doppler flowmetry probe is placed over the probe to measure the red blood cell flux. The local skin temperature is clamped or stimulated with direct heat application, and pharmacological agents are perfused through the probe to stimulate or inhibit intracellular signaling pathways in order to induce vasodilation or vasoconstriction or to interrogate mechanisms of interest (co-factors, antioxidants, etc.). The cutaneous vascular conductance is quantified, and mechanisms of endothelial dysfunction in disease states can be delineated.

作者聚焦:一种揭示心血管疾病风险人群机制的药物解剖方法 

Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation.

皮内微透析:研究人类微血管功能障碍新机制的方法

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally ...

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

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此视频中的章节

从手术标本的人类胰岛细胞激光显微切割的改进方法

瘦素受体

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

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

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

激光多普勒灌注成像在鼠标欣德利姆

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

皮内微透析:研究人类微血管功能障碍新机制的方法

皮内微透析:研究人类微血管功能障碍新机制的方法

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

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此视频中的章节

从手术标本的人类胰岛细胞激光显微切割的改进方法

瘦素受体

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

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

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

激光多普勒灌注成像在鼠标欣德利姆

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

皮内微透析:研究人类微血管功能障碍新机制的方法

皮内微透析:研究人类微血管功能障碍新机制的方法

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口