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

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

科研

医学

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

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 ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

眼底摄影作为一种方便的工具，研究微血管回应流行病学研究心血管疾病的风险因素

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

测量升主动脉刚度<em&gt;在体内</em&gt;小鼠使用超声

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

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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高含量

磷31磁共振波谱分析:一种测量工具

猪胫骨移植术后血管生成的动物模型