作者要感谢江红，博士和Deepa Parathasarthy，公共卫生，为企业发展服务的技术援助的

1。全采血车<ol><li>从人类或实验动物在NEM，/​​ EDTA含有管收集静脉血。 </li><li>立即降速血液14,300 RCF（相对离心力）7分钟准备血浆和红血细胞沉淀在台式离心机。 </li><li>准备高效液相色谱（HPLC）和化学发光检测（CLD）分析血浆样品。 高效液相色谱法:1:1血浆，涡旋离心10分钟沉淀血浆蛋白，在13200转冷甲醇的体积。高效液相色谱分析收集上清。 慢性肺病:等分样品和磺胺和升汞专门检测亚硝基垫上预温育。 </li><li>准备红细胞颗粒高效液相色谱法和CLD分析。 高效液相色谱法:添加1:4红细胞颗粒低渗裂解液中含有10毫米NEM的，2.5毫米EDTA和10毫米的铁氰化钾。涡彻底，然后加入1:1的甲醇，涡和centrif的威热10分钟，沉淀蛋白在13200转。高效液相色谱分析收集上清。 慢性肺病。加入1:4红细胞颗粒低渗裂解液中含有10 mM的EDTA，EDTA的2.5毫米和10毫米的赤血。等分样品含有亚硝基具体检测磺胺和升汞管。 </li></ol> 2。组织提取和制备<ol><li>以确定NO的代谢产物的组织水平，这是首先要收获的血液样品制备游离组织如上所述。全血交换将通过注入左心室心尖生理缓冲的地方。一旦被删除，所有的血液组织的利益，然后可以收获。 </li><li>均质组织样本，并准备高效液相色谱法和CLD分析以上所述的样品。 </li></ol> 3。血管内皮功能的主动脉环隔离组织器官浴<ol><li>小鼠将是ANEST乙醚hetized的，直到没有再回应从头到脚捏，并接受颈椎脱位，手术前。进行开胸手术暴露胸，腹主动脉。 25计注射器插入左心室心尖和灌注血液含氧克雷布斯Henseleit缓冲。 </li><li>右心房被切断，提供血液的出口。将被删除腹主动脉外膜清洗。 </li><li>戒指将被切成2毫米长的段，并安装一个四通道的组织器官浴沐浴在生理缓冲溶液（大唐720MO，公元仪器）。 </li><li>含氧，克雷布斯缓冲区（95％O 2和5％CO 2）保持在37°C，在10毫升的器官浴血管环一克的预紧被放置在每个主动脉环（最佳的血管舒缩功能的张力适当的起点在以前的实验确定）。一个八进制八通道桥（PowerLab系统）和数据采集软件（图5.2.2版本）重新用来记录所有的测力。 </li><li>环缓冲区各器官浴80分钟改为每20分钟允许平衡。平衡80分钟后，1μM的福林添加到每个亚最大收缩环。 </li><li>稳定后，将被添加到内皮细胞乙酰胆碱受体激动剂如确定没有生产和血管松弛的程度。乙酰胆碱的剂量反应后，浴池将被冲洗和recontracted然后处理与外源性一氧化氮，硝普钠，以确定平滑肌的反应，并得到一个价值100％放宽。 </li><li>洗澡可以增加NO的拾荒者，清楚地说明了NO的释放和抑制血管松弛。 </li></ol> 4。代表结果使用的ENO-20专用高效液相色谱法提供了一个易于使用的高通量方法的特异性和敏感性，亚硝酸盐和硝酸盐检测生物样品。检测和原始色谱的原理如图1所示。这种方法可用于任何生物样品的测定亚硝酸盐和硝酸盐。 CLD的NO的代谢产物的检测，需要化学derivitization步骤，确定了NO分子源。同时的氧化denitrosation和化学发光检测器还原denitrosation的实验装置如图2所示。反应釜上的权利是充满800MM在PBS pH值7.4的铁氰化钾和左侧的反应容器与碘化钾/碘醋酸减少denitrosation的（ 图2A）的混合物填充。整个安装如图2B所示。 图3说明了具体denitrosation组检测，可以检测和量化亚硝酸盐，亚硝基，亚硝胺以及亚硝基血红素产品。这种方法已经被先前descri床和验证的4,5。这些重要的生化分析，可以很容易地与对离体主动脉环的功能研究，以确定内皮细胞NO的实验动物生产。这种古典的药理实验，可以轻松，准确地评估内皮功能和生产。血管反应性的测量，如内皮细胞乙酰胆碱受体激动剂，可直接确定内皮细胞NO的生化标志物检测实验动物血液和组织中，然后可以与相关的生产。 如图4所示剂量乙酰胆碱的反应的一个典型代表。内皮功能正常的健康对照组乙酰胆碱放宽。小鼠血管内皮功能障碍（高胆固醇血症小鼠）由于减少NO生成的相同的刺激，减少松弛。 <img alt="图1" src="/files/ftp_upload/3722/3722fig1.jpg" /> 无花果余吕杏茜1。亚硝酸盐和硝酸盐的ENO-20和样品色谱检测原理。 ENO-20的亚硝酸盐和硝酸盐的检测方法示意图（上）。 （底部）10 pmol亚硝酸盐和硝酸盐的标准chromotogram注入的ENO-20（100μL亚硝酸盐和硝酸盐为100纳米的解决方案）。 1纳米为每100μL注射量的阴离子的灵敏度。没有与蛋白质或彩色物种的干扰。 <img alt="图2" src="/files/ftp_upload/3722/3722fig2.jpg" /> 图2。CLD的实验装置，用铁氰化钾还原使用碘化物/碘检测与清除一氧化氮气体的气相位检测denitrosation都氧化denitrosation。 <img alt="图3" src="/files/ftp_upload/3722/3722fig3.jpg" /> 图3（A组）化学发光法检测亚硝酸盐，RSNO，在还原denitrosation的检测RNNO样品孵育组特定的化学人的试剂。峰面积的增减允许检测亚硝酸盐和RSNOs。 （B组）化学发光法检测亚硝基血红素利用铁氰化钾的氧化denitrosation解决方案的物种。这种方法是针对无血红素产品没有RSNOs（谷胱甘肽或白蛋白，SNO），或RNNO（NO-吡咯烷N-亚硝基-白蛋白）的交叉反应。 <img alt="图4" src="/files/ftp_upload/3722/3722fig4.jpg" /> 图4。 体外器官离体主动脉环浴内皮细胞NO，然后可以用相关的生化标志物检测高效液相色谱法和CLD的生产提供了一个直接的措施。这个数字足以说明在小鼠血管内皮功能障碍的减少松弛，由于NO产量下降。

<ol>
	<li>Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+metabolism+and+breakdown.">Nitric oxide metabolism and breakdown.</a> Biochimica et Biophysica Acta. 1411, 273-289 (1999).</li><li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetycholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetycholine.</a> Nature. 288, 373-376 (1980).</li><li>Ignarro, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+factor+produced+and+released+from+artery+and+vein+is+nitric+oxide.">Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.</a> Proc. Natl. Acad Sci. U.S.A. 84, 9265-9269 (1987).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+S-,+N-,+and+heme-nitrosylation+in+biological+tissues+and+fluids:+implications+for+the+fate+of+NO+in+vivo.">Concomitant S-, N-, and heme-nitrosylation in biological tissues and fluids: implications for the fate of NO in vivo.</a> FASEB J. 16, 1775-1785 (2002).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+nitric+oxide+levels+in+the+red+cell:+validation+of+tri-iodide-based+chemiluminescence+with+acid-sulfanilamide+pretreatment.">Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.</a> J. Biol. Chem. 281, 26994-27002 (2006).</li><li>Angelo, M., Singel, D. J., Stamler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+S-nitrosothiol+(SNO)+synthase+function+of+hemoglobin+that+utilizes+nitrite+as+a+substrate.">An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 8366-8371 (2006).</li><li>Bryan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+NO+in+human+red+blood+cells:+fact+or+artifact.">Bound NO in human red blood cells: fact or artifact.</a> Nitric Oxide. 10, 221-228 (2004).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Processing+of+Nitrite+in+Hypoxia:+An+Intricate+Interplay+of+Nitric+Oxide-Generating+and+-Scavenging+Systems.">Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.</a> J. Biol. Chem. 283, 33927-33934 (2008).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic. Biol. Med. 43, 645-657 (2007).</li><li>Lundberg, J. O., Weitzberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NO+generation+from+inorganic+nitrate+and+nitrite:+Role+in+physiology,+nutrition+and+therapeutics.">NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and therapeutics.</a> Arch. Pharm. Res. 32, 1119-1126 (2009).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nat. Rev. Drug. Discov. 7, 156-157 (2008).</li></ol>

没有利益冲突的声明。

在这里介绍了有关NO的代谢产物，在多个生物车厢量化的方法将允许没有测量由血管内皮细胞NO的功能，可与相关的健康和疾病的生物学指纹。为适应高吞吐量的潜力，这些方法需要简单的样品制备。这些分子的相对数量，可以帮助了解NO的生产和其代谢的命运，在一些疾病，甚至从人类患者的串行样品实验模型。检测没有基于生物标志物和许多分析方法，人为地制造一些样品制备过程中，这些产品也有很多陷阱。仔细核算生物样品中的亚硝酸盐是至关重要的，由于其反应与半胱氨酸硫醇6 artifactual形成亚硝基7。基于CLD的亚硝酸盐在厌氧条件下的具体检测方法，导致在由于特定的亚硝酸盐还原酶酶的活动在不同的生物车厢8一致的结果。这里介绍的所有方法以及记录和验证，并需要考虑的步骤，以消除或减少文物9。我们将描述必要的关键步骤，以防止文物。这个NO的代谢产物多室检查将帮助确定相关的生物标志物，可能在人类诊断或预后的实用工具，然后将允许可以在诊所或标准的实验室分析用标准化的方法发展。最近承认一个人的氮循环，即硝酸盐和亚硝酸盐减少无由硝酸10,11 enterosalivary流通到现在开辟了潜在的使用作为一个没有在某些疾病状态的潜在生物标志物的唾液。迄今没有状态是不标准的血液用于诊断患者的常规化学的一部分。日令人震惊没有许多疾病进程​​的关键性质。最终，确定这些不相关的生物标志物的水平易于使用，并可能快速床边照顾点经过充分验证的检测将是一个真正的证明了他们在分子医学的作用。它是在该领域的集中和联合的努力，以验证和发展标准和准确的检测，确定无状态动物模型验证人类在这个时刻谨慎。在这个协议中所述的方法将允许他人迅速采取共识，这样的测量方法。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> N - 乙基</td><td> Thermo Scientific的</td><td> 23030 </td></tr><tr><td> EDTA的</td><td> Sigma-Aldrich公司</td><td> E7889 </td></tr><tr><td>铁氰化钾</td><td> Fluka公司</td><td> 60299 </td></tr><tr><td>高效液相色谱法</td><td> eicom公司</td><td>的ENO-20 </td></tr><tr><td>自动进样器</td><td>奥尔科特</td><td></td></tr><tr><td>大唐肌动描记</td><td>公元仪器</td><td></td></tr><tr><td> PowerLab系统</td><td>公元仪器</td><td></td></tr><tr><td>化学发光</td><td> EcoPhysics </td><td> CLD的88Y </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5415D </td></tr><tr><td>乙酰胆碱</td><td> Sigma-Aldrich公司</td><td> A6625 </td></tr><tr><td> ，R-（ - ）苯肾上腺素</td><td> Sigma-Aldrich公司</td><td> P6126 </td></tr></tbody></table>

analytical techniques for assaying nitric oxide bioactivity

一氧化氮（NO）是一种双原子自由基是极短的住在生物系统（血液循环小于1秒）1。 NO可能被认为是最重要的信号分子在我们的身体产生的，规范的基本职能，包括但不限于调节血压，免疫反应和神经通讯。因此，其准确的检测和生物样品的定量是在健康和疾病的认识没有任何作用的关键。与这种无生理半衰期短，检测NO的生物化学反应产物的替代战略已经制定出来。有关NO的代谢产物在多个生物舱的量化提供有价值的信息，对于体内没有生产，生物利用度和代谢。简单采样，如血液或血浆的单室可能并不总是准确的评估提供了一个全波DY没有地位，特别是在组织。的能力比较与选择组织实验动物的血液，将有助于弥合尽可能NO的生物标志物在健康和疾病的诊断和预后实用的基础科学和临床医学之间的差距。因此，血浆或血液利益的具体组织无状态的推断已经不再是一个有效的方法。因此，方法不断得到发展和验证允许NO和NO相关产品/多个车厢在实验动物体内的代谢产物的检测和定量。建立了从生产模式，通过NO合成酶的激活可溶性鸟苷酸环化酶（sGC）的最终氧化为亚硝酸盐（NO 2 - ）和硝酸盐（NO - 3）NO的生物化学只可代表体内 NO的影响的一部分。与蛋白质巯基，二级胺，金属NO和NO源性代谢物形成的S-nitrosothi的相互作用OLS（RSNOs），N-亚硝胺（RNNOs），亚硝基血红素分别代表NO cGMP独立的作用，并有可能通过NO激活SGC一样重要生理。一个真正了解NO在生理来自体内实验，同时采样多个车厢。一氧化氮（NO）的方法，是一个复杂的，往往是混淆不清的科学和关于NO的生物化学许多辩论和讨论的焦点。阐明新的机制和我们的能力，具体涉及无铰链的信号通路，选择性和灵敏的检测和量化NO和所有相关的NO产品和复杂的生物样品中的代谢产物。在这里，我们提出了一个为亚硝酸盐和硝酸盐的快速，灵敏的高效液相色谱分析方法以及使用与化学derivitazation 在体外臭氧的化学发光法测定分子源NO以及体外与生物样品的检测不含器官浴myography。

Watch this Scientific Journal Video about Analytical Techniques for Assaying Nitric Oxide Bioactivity at JoVE.com

Analytical Techniques for Assaying Nitric Oxide Bioactivity

内源性一氧化氮（NO）的生产，调节多种生物功能。人们越来越清楚NO基于信号中断或失调，在涉及人类的许多疾病。量化方法有关NO的代谢产物可能为人类疾病提供新的诊断或预后标志物。

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

analytical-techniques-for-assaying-nitric-oxide-bioactivity

Nanyang Technological University

Research

JoVE Journal

Medicine

17.9K 次观看.  University of Texas Health Science Center at Houston. 内源性一氧化氮（NO）的生产，调节多种生物功能。人们越来越清楚NO基于信号中断或失调，在涉及人类的许多疾病。量化方法有关NO的代谢产物可能为人类疾病提供新的诊断或预后标志物。

视频: Analytical Techniques for Assaying Nitric Oxide Bioactivity

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th联合切除术，高盐饮食和一氧化氮合酶抑制剂诱导Lewis大鼠慢性肾脏病

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

科研

医学

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

技术处理眼睛的视网膜假体植入局部组织病理学分析

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

对于检查纤维蛋白凝块结构在正常和疾病状态的实验和成像技术

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

超超磁氧化铁纳米探针在肺外结核检测的合成、表征及应用

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

心脏磁共振评估疑似心脏血栓:常规和新兴技术

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

近红外光免疫疗法为小鼠模型的胸膜传播

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

一种新型吸入面膜系统，可在自发呼吸受试者中提供高浓度的一氧化氮气体

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...