这项研究是由美国国立卫生研究院/ NIBIB R21EB008166支持DL和NIA的AG028455和NIA的粉喷桩AG022385部分。我们感谢提供他们D282荷瘤小鼠的弗雷德哈钦森癌症研究中心的詹姆斯奥尔森博士。

第1部分：安全磁铁 MRI和MRS使用一个强大的磁场，需要格外小心。例如，仪器，我们目前的工作中使用的4.7 T磁场，地球磁场的约90000倍。高磁场始终是即使不被使用的磁共振扫描仪。任何金属物体接触到这样一个高磁场的强烈和迅速将磁铁吸引。这是极其危险的，如果一个实验题目或运营商是在位于飞入磁铁的金属对象的弹丸路径。因此，进行致辞实验的人员应小心删除任何金属物体进入仪器接近前从他们的服装，并保持周围的环境，这样的对象。磁铁安全的更详细的信息出现在文献 1以下网页<a href="http://www.imrser.org/">：</a> ​​http://www.imrser.org/ 。金属材料的存在，不仅可以造成上述安全问题，但诱导影像/光谱文物与实验结果的干扰。在附近成像物体附近或内部的金属材料存在可以改变磁场，使获得的图像生成的工件或扩大的谱线宽度。 因此，外磁铁离开钱包，钥匙，钢笔等，如果你处理的动物致辞程序。 第2部分： 在小鼠体内磁共振成像在水平孔磁铁动物的准备MRI <ol><li>所有动物的程序应经体制的动物护理和使用委员会（IACUC）前执行任何类型的动物处理。 </li><li>我们使用汽化异氟醚麻醉用于核磁共振成像实验的动物。 avertin（2,2,2 - tribromoethanol或简称TBE）和氯胺酮和甲苯噻嗪的鸡尾酒，如其他麻醉药，麻醉动物可以实现。每个麻醉剂用量的信息是发现于表1。 </li><li>线塑料支持的吸水垫（蓝色的垫子或卡盘）的感应室。将鼠标（或多个小鼠多鼠标成像），在诱导室。 </li><li>调整异氟醚蒸发器流量计0.8 1.5 L / min的。然后调整至4％的异氟醚蒸发器，约2 3分钟。 </li><li>达到手术的麻醉平面（即没有脚趾捏响应）后，地方上的动物持有人的鼠标，它的鼻子插入一个鼻锥（或面罩）。头限位可用于头成像的机构持有人可使用全身显像。 </li><li>动物持有人使用，以防止潜在的运动：有几种类型的商业持有人。此外，定制设计的持有人可以编造的实验装置，以适应任何特殊要求。定制设计的持有人，确保只使用非磁性物质。 </li><li>成像期间，调整流量计0.4 0.8毫升/分钟，减少到1.2〜1.5％的异氟醚蒸发器。过期的天然气来自鼠标鼻锥是由泵收集到一个内部真空中删除。 </li><li>动物的眼睛保持湿润无菌眼润滑软膏。将保持在35-37 ° C，在一个温暖的循环水系统内的实验动物。可用于其他类型的热源，如加热垫，暖空气吹入的射频（RF）线圈。 </li><li>动物监测系统是监测体温，呼吸/心脏周期，并与影像收购同步呼吸/心脏门控到位。 </li><li>琼脂糖标准样品被放在旁边的动物监察突然信号变化。本标准琼脂样品是多层面和多时间点成像特别有用。当一个意外的信号变化是从采集到的图像切片检测的，意外的信号变化的切片可以消除的。此外，在后的图像分析标准样品的信号变化的基础上，其信号强度可以调整。 </li><li>安全放在动物的人，动物和监视组件后，动物的人，在一个射频线圈的中心位置。 </li><li>移动射频线圈，磁铁的房间，并插入到系统内的磁铁放置温水循环射频线圈。图2显示，从磁铁的前面观察核磁共振成像扫描仪的几个组件孔。 </li></ol>磁共振成像实验<ol><li>调谐射频线圈的共振频率的1 H和线圈的特性阻抗相匹配到50欧姆的磁共振扫描仪中使用调整面板。这是为了达到最佳的信号接收条件。大多数人类的核磁共振成像扫描仪不需要一个单独的进程中调整/匹配除刘健程序。 </li><li>进行一个匀场的过程中，使用单脉冲序列。 MR信号依赖于环境的磁场均匀。垫补过程使该地区的利益，尽可能同质的磁场。每个磁共振扫描仪，有它自己的方式来执行的垫补过程中，包括自动快速快速地图和梯度垫补垫补过程。 </li><li>优化射频脉冲，通过最大化的一个三维图像轮廓。射频脉冲的权力，可以排列，同时保持恒定的脉冲长度和一个足够长的TR（回收拖延），大约是3 - 5倍的组织T 1 。 </li><li>沿三个正交方向创建轴向，冠状面和矢状面图像采集球探图像。一个快速的图像采集序列（即梯度回波或快速自旋回波成像序列）可以用来获得球探图像。采集到的图像将被用于计划与成像平面的决心的实际成像。 </li><li>更改自旋回波序列。选择适当的序列参数：TR（回收拖延）应三至五倍的组织 T 1，以获得充分的放松，如质子密度或 T 2加权图像图像。 TE（回波时间）之间的首个射频脉冲和回波信号的中心的持续时间。可以选择的TE​​值取决于图像的对比度，如表2总结。图2显示了与T 1，T 2和T 2 *不同的松弛效应获得了一个在它的后面的移植瘤的裸鼠体内图像。 </li><li> T 2的测量可以使用multiecho成像或单一回波成像与多个TE值。 </li><li> MRI / MRS在体内实验后，应监测动物的整个恢复过程中。磁共振成像后，将取出的动物射频线圈和监测，以确保完全恢复时返回到笼。在麻醉小鼠的快速热损失。保持温暖的动物，其中包括用纱布垫或毛巾和/或提供热源，直到动物从麻醉中恢复。 </li></ol>图像处理中的<ol><li>回顾收购致辞控制台上的图像和选定的数据传输到后处理的计算机。 </li><li>我们通常使用ImageJ（ <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>分析图像）。图像分析，包括图像缩放/过滤，计算T 1，T 2和扩散，肿瘤体积测量及肿瘤的分割。 </li></ol>第3部分。在一个垂直的玻尔磁体内小鼠后肢骨骼肌刘健袖口建设诱发可逆性缺血<ol><li>用PVC管件，约5-7毫米，宽12-15毫米的ID开始。钻了一个小洞，通过墙上的这块和线程。 </li><li>剪下一块的气球，所以它是开放两端（氦的质量气球最好的工作）。通过PVC片插入件和包装左右带子两端外墙上的PVC片。 </li><li>使用收缩包装密封气球两端管周围。你应该有一个具有坚实的外墙和一个充气的内壁袖口。 </li><li>剪下的面积收缩包装和气球的螺纹孔周围，照顾孔的PVC片的边缘之间留下大量的材料。线程A非铁金属（如铜）1.5厘米成孔在PVC片。这可以让你充气袖带。 5分钟的环氧密封区。 </li><li>修正了在袖口旁边的射频线圈在刘健探头并连接到一个外部的血压计。 </li></ol>自定义呼吸监视器<ol><li>我们使用一个自定义的呼吸监测是要受限制的空间和访问磁铁的孔兼容。一些商业模式也可提供。 </li><li>一个小气球，以铁的耐拉伸管馈入探针年底。 </li><li>连接管压力传感器的另一端。 </li><li>确保线路和气球的气泡。气泡会衰减信号压缩鼠标身体因呼吸运动造成的气球。 </li></ol>鼠标定位在刘健探头<ol><li> 5％Avertin（0.010毫升/克体重）麻醉鼠标。 </li><li>达到手术的麻醉平面后，位置刘健探头鼠标，鼠标支持鼠标放置在它的后面。鼠标放在腹部充满液体的气球，并确保在代替鼠标支持带。 </li><li>刘健探头位置鼠标和身体的支持。 </li><li>拉出之一后肢缺血的袖口和刘健线圈。腿应集中在线圈亩CH尽可能。这种安排使鼠体定位在一个垂直的孔磁铁的水平（图3）。 </li><li>修正盘带脚刚性支持腿到位。 </li><li>在区域外的线圈采样的后肢皮下放置热电偶。 </li><li>浸湿的眼睛，用眼润滑软膏，以防止眼睛干燥。包括鼠标的眼睛和脸从墙上探头，以防止摩擦或刺激。 </li><li>根据实验的具体需求，可以添加其他监控探头。 </li></ol>设置刘健实验<ol><li>刘健探头连接气流加热元件。 </li><li>设置可变温度控制单元VNMR软件腿温度保持在35-37 ° C。 </li><li>调整线圈的频率和匹配阻抗在1 H和31个P共振刘健软件的使用调整面板。 </li><li>调整垫片电路优化B1磁场的均匀性，在使用1 H光谱利益的地区。 </li><li>切换到31个P频率来确定射频脉冲宽度产量最大的信号从一个自由感应衰减（FID）（90 °）。 </li><li>收集高信号噪音完全放松谱（FRS），以确定完全放松的条件下无机磷的比率 （P I）和磷酸肌酸（PCr）的ATP。这些光谱收集，使用90 °的时间与时间之间的FID收购（TR）PCR 的 T 1（20秒7 T）的约5倍。这些将被用于定量PCR和P 我先生谱水平。 </li></ol>缺血性实验<ol><li>一个简单的缺血性扰动允许的休息，最大的线粒体ATP生产的决心，通过测量在磷酸变化和紧接缺血。 </li><li>设置阵列来收集多光谱，采用45 °的脉冲宽度（即0.5 × 90 °的时间）和0.5X的TR的T 1（1〜1.5秒） 。我们通常收集4 FID的每一个约6秒的时间分辨率光谱（VNMR软件的平均数目（NA）= 4）。这个时间分辨率是足够准确地确定休息和最大的线粒体ATP生产。 </li><li>收集约5分钟的休息光谱。 </li><li>诱导缺血10-12分钟到270-300毫米汞柱袖带充气。 </li><li>松开袖口和恢复谱收集5分钟。 </li><li>取出磁体和探头，探头鼠标。让鼠标在适当的条件下从麻醉中恢复。在随后的日子里，可反复实验。经过最后的光谱实验小鼠腿部肌肉在液态氮将被删除，立即冻结钳位ATP浓度的高效液相色谱分析。 </li></ol>数据分析<ol><li>数据进行离线分析使用NMR谱的谱分析的几个方案之一。我们的实验室通常使用合适的标准2和jMRUI（http://www.mrui.uab.es/mrui/mrui_Overview.shtml）量化峰面积。 </li><li> PCR初步的故障率在缺血常氧条件下线粒体ATP生产的措施，在3-5几篇论文描述。 PCR的回收率可用于确定线粒体ATP生产的最大容量的基础上的方法 ，以前4,6介绍。 </li></ol> <a href="https://www.jove.com/files/ftp_upload/1592/1592fig1.jpg"><img border="0" src="/files/ftp_upload/1592/1592fig1tmb.jpg" alt="图1" /></a> 从磁铁的前端看在体内MRI设置图1。孔。设置包括一个射频线圈，动物变暖系统（或温循环水系统）和梯度插入。所有这些组件将被插入到一个水平磁铁。温水中加热磁体室外面的一个蓄水池，通过聚乙烯管（绿色磁带）的动物变暖系统介绍。在汽缸内循环后，水是从拉暖系统水库被加热。一个异氟醚管和真空集热管是用来麻醉在核磁共振实验小鼠。 <a href="https://www.jove.com/files/ftp_upload/1592/1592fig2.jpg"><img border="0" src="/files/ftp_upload/1592/1592fig2tmb.jpg" alt="图2" /></a> 图2在体内与裸小鼠移植瘤在它的后面不同的松弛效应（箭头）（D282肿瘤）图像：A. T 1。称重形象“（TR / TE = 500/14.2ms ）。 B. T 2重形象“（TR / TE = 2s/40ms） 。这两种T 1和T 2加权图像获得自旋回波序列。 C. T 2的地图处理与图像DIF收购的4套ferent TE值从20到80毫秒。 D. T 2 *称重（TR / TE = 180/7.39ms，翻转角= 20度）收购梯度回波序列。 35 × 35毫米2场是所有MR图像。 <a href="https://www.jove.com/files/ftp_upload/1592/1592fig3.jpg"><img border="0" src="/files/ftp_upload/1592/1592fig3tmb.jpg" alt="图3" /></a> 图3。插图鼠标定位在水平身体与腿架固定在射频线圈。 <a href="https://www.jove.com/files/ftp_upload/1592/1592fig4.jpg"><img border="0" src="/files/ftp_upload/1592/1592fig4tmb.jpg" alt="图4" /></a> 图4 31个P光谱在体内通过一个缺血再灌注周期。数据被收集在一个7 T垂直孔磁铁和20赫兹指数滤波器扩大线。 TR = 1，NA = 4，每20谱绘制。 表1。鼠标MRI /刘健麻醉剂用量。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 动物 物种 </td><td> 麻醉剂 代理 </td><td> 剂量 （毫克/公斤为注射） </td><td> 路线 </td></tr><tr><td>鼠标</td><td>异氟醚气体</td><td> 2-3分钟（感应）的4.0％，1.2至1.5％连续（维护） </td><td>吸入通过鼻锥</td></tr><tr><td>鼠标</td><td> Avertin </td><td> 5％，10ml/kg体重</td><td>腹腔（IP） </td></tr><tr><td>鼠标</td><td>氯胺酮/甲苯​​噻嗪</td><td> 100毫克/公斤和10毫克/公斤</td><td>知识产权</td></tr></tbody></table> 表2。放松比重的图像<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 图像加权 </td><td> TR（回收延迟） </td><td> TE（回波时间） </td></tr><tr><td> T1 </td><td>短（T1短） </td><td>短（短于T2） </td></tr><tr><td> T2 </td><td>长（3〜5倍的T1） </td><td>长（约T2） </td></tr><tr><td> PD（质子密度） </td><td>长（3〜5倍的T1） </td><td>短（短于T2） </td></tr></tbody></table> T1：自旋晶格弛豫时间（或纵向弛豫） T2：自旋自旋弛豫时间（或横向弛豫）

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调整/匹配和垫补收购前的步骤是至关重​​要的，以获得高空间分辨率和高的信号噪声比（SNR）。同样重要的是监测动物与动物监测系统，以保持在一个稳定的信号采集的生理状态的动物，符合人性化的动物处理，以防止任何潜在的artifactual测量条件。在协议解释的程序，可以进行修改，以获取更多的信息，包括在体内的扩散，灌注和血流成像和本地化光谱。所有的动物准备工作应该是相似的，?...

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<td>Inova 200 MR scanner/4.7 T</td>
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<td>Varian/Bruker</td>
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<td>Used for mouse MRI</td>
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<td>Inova 300 NMR spectrometer/7 T</td>
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<td>Varian/Oxford</td>
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<td>Used for MRS of mouse skeletal muscle</td>
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noninvasive in vivo small animal mri and mrs: basic experimental procedures

小动物磁共振（MR）的研究已成为现代生物医学研究，由于其非侵入性的性质和它提供了丰富的生物信息的重要元素。议员并不需要任何电离辐射，非侵入性，可以提供更高的分辨率和更好的信号噪声比在其他断层或光谱方式相比。在这个协议中，我们将重点放在小动物磁共振成像和磁共振波谱（MRI / MRS）的非侵入性收购放松加权<sup&gt; 1</sup&gt; H图像的​​鼠标，并获得<sup&gt; 31</sup&gt; P光谱小鼠肌肉。这项工作并不试图涵盖所有方面的小动物MRI /刘健，而是引入鼠标MRI /刘健实验的基本程序。这项工作的主要目标是告知研究人员的基本程序<em&gt;在体内</em&gt;致辞实验的小动物。我们的目标是提供一个更好地了解基本的实验程序，允许研究人员的新致辞领域，以更好地规划，使议员和非议员的程序无缝集成的研究非议员组成部分。

Watch this Scientific Journal Video about Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures at JoVE.com

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

这项工作描述无创性的小动物MRI和MRS的基本程序<em&gt;在体内</em&gt;。

无创在体内小动物的MRI和MRS：基本实验程序

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

noninvasive-vivo-small-animal-mri-mrs-basic-experimental

Research

JoVE Journal

Biology

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

16.0K 次观看.  University of Washington. 这项工作描述无创性的小动物MRI和MRS的基本程序在体内。

视频: Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

科研

生物学

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

合成的<em&gt;在体内</em&gt; MRI检测细胞凋亡的探讨

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

鸡寄生虫的成核和活体繁殖

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

小尺寸远程模型日本美达卡（奥里齐亚斯拉蒂普斯）的果纳切除术和血液采样程序

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...