这项工作是由美国国立卫生研究院（U54CA143837），美国国立卫生研究院MD安德森癌症中心支持资助（CA016672），在V基金会（SAC），以及从Kanzius研究基金会（国资委，伊利，宾夕法尼亚州）不受限制的研究经费。我们感谢和Kristine从灰肿瘤外科部，MD安德森癌症中心，对行政协助。

一个完整的实验概述如图1所示。 进一步的细节被描述在下面的步骤1月3日。 1。 NP胶体评估射频加热：金纳米粒子为例。 <ol><li>在一般情况下，对于每个样品NP正在研究中，首先通过离心过滤洗涤样品数次，用去离子（DI）水，以去除背景离子和污染物。所有离子和污染物将被从什么时候被洗出液也有类似的射频加热速率（HRS）为去离子水在金纳米粒子悬浮液中除去。此纯化过程还允许粒子以获得更高的浓度。值得注意的是，尽管在本例中使用金纳米粒子，基本原理可以应用到其它的NP材料。 </li><li>作为一个例子，净​​化的500ml瓶的直径为5nm市售的金纳米粒子，然后使他们受到电连接的一个13.56MHz的射频场ELD强度90千伏/米。 <ol><li>拿〜125毫升从六个50 kDa的离心过滤管之间的股票金纳米粒子溶液和分裂。离心机在3000转每分钟。 2分钟5秒。更多原液过滤除去缓冲和填充的过滤器。重复，直到所有500毫升已被过滤。 </li><li>更换过滤缓冲液DI水相似的体积和重复约8倍（或直到滤波后的RF缓冲器的HR等同于DI水中）。注意，紫外 - 可见分析也可以用来监视污染物的吸收峰。一旦缓冲区污染物已完全清除，吸管约0.5毫升去离子水到每个过滤器和通过反复吹打重悬金纳米粒子。这应该完全从过滤器中删除金纳米粒子，并允许充分重悬。结合所有六个悬浮成一个15毫升的Eppendorf管中。 </li><li>一旦金纳米粒子被提纯和浓缩，用ICP-OES和/或ICP-MS，UV-vis和Zeta电位上concentratio数据分析样本n和NP稳定，分别为。 SEM和/或TEM分析，也可用于获得形态学数据。对这些技术的详细的样品制备可以在文献4中找到。 </li></ol></li><li>通过在先前的研究中8所述的Kanzius射频系统或该系统的派生，放置一个1.3毫升圆柱形石英比色皿，使空气中的射频电场（无样品存在）将〜90千伏/米的反应杯内。对于一个标准的盐水样品（0.9％NaCl）中的电场将减少到〜1.1千伏/米。这些是用来允许在不同系统之间进行的比较的近似条件。 <ol><li>吸移管130的纯化的金纳米粒子胶体到石英比色皿1000毫克/升样品的溶液和该引入到RF场。这可以通过使用一个定制的聚四氟乙烯样品架来完成。样品暴露于RF场为一个周期120秒或直至样品达到70℃，以防止电弧或快速沸腾。钙利用红外相机及相关软件pture热成像数据（以及控制区）。重复此步骤三次。 </li><li>通过另一个50 kDa的离心过滤器过滤样品，从去离子水缓冲液中提取的金纳米粒子。缓冲区重新暴露于RF场，再次三次。在金纳米粒子胶体和背景DI水缓冲器之间的HR的差异决定了HR由于金纳米粒子本身。预计取得的HR〜0.3°C /秒和0.05℃/秒，得到〜0.25℃/秒的金纳米粒子相关的人力资源。重悬从过滤器的剩余金纳米粒子130毫升水中的体外/体内实验。 </li></ol></li></ol> 2。纳米粒子辅助射频热疗诱导： 体外研究<ol><li>这些体外研究可以应用于任何类型的形成2D单层癌细胞类型。在这个实验中使用派生的Hep3B细胞肝癌。 <ol><li>板〜50,000 CELLS在前面3口井12孔板用1毫升生长培养基中。重复此6倍（使用三个板块为NP的研究和三大板块作为对照）。孵化在37.5℃，引入纳米颗粒前24小时。使用生物安全柜紫外线灯照射5分钟消毒粒子第一。 </li><li>在每孔中介绍加入0.1ml 1000 mg / L的金纳米粒子的溶液，留下另外24小时。加入0.1毫升的水成三个控制细胞板的每个孔中，并放置24小时。 </li><li>后24小时已经过去了，吸出细胞培养基，用PBS洗涤以除去任何表面结合的金纳米粒子。更换细胞培养基。细胞现在已经准备好为RF暴露。 </li></ol></li><li>将射频场中的每个12孔细胞组。等到细胞已经冷却到31°C。打开RF发生器和暴露为3.5分钟。细胞培养基的最终温度为〜37℃。关闭RF场。取下电池，并将其放置在孵化器进行分析前24小时。 <ol> &lt;LI&gt;从孵化器和吸细胞介质中删除单元格。加1.6毫升细胞培养基到每个孔中，以及在0.4毫升MTT试剂。孵育细胞4小时。吸媒体和替换用2毫升二甲基亚砜（DMSO）的。将电池板的长凳上摇臂，离开10分钟，使DMSO溶解的MTT试剂。最后，用酶标仪如SPECTROstar纳米板读取器移液管加入100μl每孔加入到96孔板中并光学读取以及在570nm处。 </li></ol></li></ol> 3。纳米粒子辅助的射频诱导热疗： 体内研究<ol><li>这些体内研究可以应用于任何类型的癌症，形成实体瘤中原位或异位小鼠模型。本实验采用的Hep3B肝癌细胞在异位瘤BALB-C裸鼠模型。 </li><li>注：所有体内实验均符合所有相关指引，法规和监管机构执行。此外，得克萨斯大学MD安德森癌症中心实验动物管理和使用委员会（IACUC）的指导和批准下进行演示的协议。 <ol><li>生长细胞的适当数量（〜100 k）在第一个组织培养瓶用适当的生长培养基中。孵育在37℃的培养箱中5％CO 2的细胞培养物的持续时间。 </li><li>处理细胞，用胰蛋白酶（从烧瓶中分离），并产生2百万个细胞每25微升的溶液中。添加基质胶的等量（冰），调匀，以准备最后的注射液。注入此溶液到鼠标的背面所需的位置，然后等待适当的时间量对肿瘤生长到所需的大小（2-4周对大多数细胞）。 RF暴露之前，BALB-c裸鼠应承担固体异位瘤0.5-1厘米直径。 </li></ol></li><li><ol><li>制备的小鼠中使用（在这种情况下的BALB-C裸鼠）由ANAEsthetizing他们用氯胺酮和甲苯噻嗪通过IP注射的溶液。当小鼠入睡，让他们在一个温度控制腔在37°C 20只小鼠中总将是需要的，所有的轴承同样大小的肿瘤。十只小鼠将与结合和未经金纳米粒子用于（后者仅PBS注射）而剩余的10只小鼠将射频曝光和未射频暴露的对照组之间进行分割：两组无金纳米粒子射入。 </li><li>一旦正确麻醉，注入纳米金直接用1毫升注射器用27号针头的肿瘤。在金纳米粒子的溶液应该是在200毫克/升的金浓度在0.1ml PBS中。注射后，用棉签手术吸收血液和擦拭注射部位用酒精擦拭。 </li><li>然后挂接鼠标放在射频发生器的接收头进行处理。鼠标必须定位，以使所述肿瘤是最接近于传输头。屏蔽并非要处理的区域，以及敏感略去，如眼睛，耳朵，脚趾和地区，铜带。可以肯定的是，这样没有电荷积聚时铜带适当接触接地平面。另外，在曝光区域必须具有至少1cm大于所需治疗部位的大小的间隙。 </li><li>位置红外热成像摄像机，使肿瘤及其治疗区域是可见的。打开射频5分钟。记录所产生的温度曲线。如果治疗达到立即温度高于42℃停止治疗。 </li></ol></li><li><ol><li>经过RF暴露恢复小鼠从麻醉中一个温暖的室内，直到他们是有意识的。 </li><li>重复实验，相同的小鼠48小时后（步骤3.3.1 - 3.4.1）。 </li><li>实验结束后，安乐死小鼠按照机构的协议和程序。记录肿瘤的重量。组织学分析解决使用福尔马林的肿瘤和它们嵌入在石蜡。肿瘤切片通常机智染色ħ苏木精伊红和相关的衡量治疗效果， 如 Ki-67的目标，裂解的caspase-3 等 。 </li></ol></li></ol>

<ol>
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我们什么都没有透露。

这些协议允许实验者充分分析的程度的纳米材料（在这种情况下金纳米粒子）可以增加射频诱导的高热用于癌症治疗。第一个协议，专门处理与分析产热量从高度集中及纯化的金纳米粒子样品。尽管其它研究小组报道的产热主要从该金纳米粒子悬浮于而不是金纳米粒子本身9-11的缓冲器，它们的射频系统中使用的较低的金纳米粒子的浓度，直径&gt; 10nm时，以及具有较低的RF工作功率电电场?...

射频能量的生物组织（由于其固有的介电常数）的吸收导致在升高组织温度作为时间的函数，这最终导致通过热疗细胞死亡。据推测，癌症热疗可通过使用靶向的纳米材料内化的癌细胞内，并作为射频热换能器，使相邻的健康，正常细胞完整的优化。有几份报告已经表明，各种纳米粒子可作为有效的射频热源，其帮助癌症坏死1-4。 在这些方面，金粒子（金纳米粒子）3-5，碳纳米管1，和量子点6,7已经表现出令人兴奋的特性在体外和体内射频实验时使用。虽然当暴露在射频领域的这些纳米粒子的加热机制的确切性质仍存在争议，一系列用金纳米粒子基础实验寄予了很大意义上都NP大小和聚集状态。它表明，直径&lt;10nm的金纳米粒子仅当暴露于射频场8将加热。此外，当金纳米粒子聚集该加热机构被显著衰减。这种聚合条件也有效的体外模型放置在endolysomal细胞车厢内优化金纳米粒子的胶体稳定性的有效射频治疗4中的重要性。然而，用于收集和评估该数据的技术和实验原理可能会产生问题，特别是在从NP胶体验证射频热访问的情况下。 一些报告显示，背景离子悬浮液，该纳米颗粒被悬浮在焦耳加热可以是射频产热的主要来源，而不是粒子本身9-12。尽管我们最近的一篇论文8已经验证吨他使​​用RF相互作用从直径小于10纳米的金纳米粒子产生热量，我们的目标是在这篇文章中详细描述了这些协议。 我们还证明了协议和评估金纳米粒子作为体外和体内实验为肝癌模型温热热剂中均有效性所需要的技术。虽然我们主要集中于柠檬酸封端的纳米金胶体简单，相同的技术可以被应用到其他金纳米粒子的杂交如抗体和化疗结合的复合物。通过遵循这些原则，实验者应该希望能够快速评估对于任何纳米材料是一种有效的射频感应热温热剂的潜力。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>500 ml gold nanoparticles (5 nm)</td>
			<td>Ted Pella, INC</td>
			<td>15702-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)</td>
			<td>Millipore</td>
			<td>UFC805024/UFC910096</td>
			<td>(4 ml and 15 ml volumes)</td>
		</tr>
		<tr>
			<td>MEM X1 Cell Culture Media</td>
			<td>Cellgro</td>
			<td>10-101-CV</td>
			<td>(add extra nutrients as necessary)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper Tape</td>
			<td>Ted Pella</td>
			<td>16072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Kanzius RF System (13.56 MHZ)</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR Camera</td>
			<td>FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)</td>
			<td>Contact FLIR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.3 ml Quartz Cuvette</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon Sample holder with Rotary Stage</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPECTROstar Nano Microplate reader</td>
			<td>BGM Labtech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Applied Nanofluorescence, Houston, TX)</td>
			<td>NS1 NanoSpectralyzer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ICP-OES</td>
			<td>PerkinElmer</td>
			<td>Optima 4300 DV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern</td>
			<td>Zen 3600 Zetasizer</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

protocols for assessing radiofrequency interactions with gold nanoparticles and biological systems for non-invasive hyperthermia cancer therapy

癌症疗法的毒性较小和侵入比现有的同行是非常可取的。使用射频电领域的深入渗透到体内，引起毒性最小的，目前正在研究作为非侵入性癌症治疗的可行手段。可以预想到的RF能量与内在化纳米粒子（纳米粒子）的相互作用可以解放热，然后可以引起细胞的过热（热疗），最终结束于细胞坏死。 在非生物系统的情况下，我们提出有关量化由高浓度的NP胶体释放的热​​量详细的协议。对于生物系统，在体外实验的情况下，我们描述了必须坚持，才能有效地揭露癌细胞射频能量而不散装媒体加热工件显著模糊数据的技术和条件。最后，我们给出了一个详细的方法f或体内小鼠模型异位肝癌肿瘤。

1。评估的NP胶体的射频加热：纳米金作为例子。 下一节1.1后 - 1.2.3期望有5 nm和10 nm的金纳米粒子的直径高度集中，稳定，纯净的解决方案。从500毫升如购买的原液，期望获得至少为4毫升溶液在1000毫克/升的浓度该金纳米粒子，并在此浓度的背景DI水缓冲溶液之间的HR的差值应为〜0.25℃/秒和0.1℃/秒为5 nm和10 nm的金纳米粒子，分别为显示在图2。 2?...

Watch this Scientific Journal Video about Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy at JoVE.com

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy | Medicine | Video

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

我们描述了用于研究的13.56MHz的射频（RF）的相互作用的协议电领域，在这两种非生物和生物系统（ 体外 / 体内 ）纳米金胶体。这些相互作​​用正在研究用于癌症治疗的应用。

协议的射频评估与互动金纳米粒子和生物系统的非侵入性癌症热疗治疗

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

protocols-for-assessing-radiofrequency-interactions-with-gold

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10.7K 次观看.  University of Texas M.D. Anderson Cancer Center. 我们描述了用于研究的13.56MHz的射频（RF）的相互作用的协议电领域，在这两种非生物和生物系统（ 体外 / 体内 ）纳米金胶体。这些相互作​​用正在研究用于癌症治疗的应用。 

视频: Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

科研

医学

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

纵向，非侵入性的移植到眼睛的前房<em在体内</em&gt;单细胞分辨率的实时成像

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.