研究在我的实验室的支持，生物学启动资金的WKU部，WKU研究基金会RCAP，我承认＃11-8032，并通过从全国学院普通医学科学研究所父母捐款资助，KBRIN-AREA补助生在奖号5P20GM103436-13。我还要感谢田旭中博士实验室的我被介绍给这个技术和雷蒙德Pagliarini博士谁首先建立这种技术在许实验室。

1。诱导良性非侵入性肿瘤<ol><li>使用表2中列出的股票为良性肿瘤的诱导。 </li><li>准备一个起始培养以下 ​​基因型的“测试仪”股票：Y，W，EY-FLP1;浴池- GAL80，FRT40A; Act5C&gt; Y +&gt; Gal4的，UAS-GFP </li><li>准备一个起始培养的“测试”股票下列基因型：W; FRT40A，UAS-V12的Ras / CYO </li><li>收集来自“测试仪”股票10只雌性童女和十男性从“测试”的股票。 </li><li>放置他们两个在一个小瓶飞食品跨越从“测试仪”和“测试”股票的男性和女性。 </li><li>将交叉于25℃培养箱中培养，并让雄性和雌性交配和产卵24-48小时。 </li><li>检查小瓶足够卵沉积并为第一龄幼虫确保培养物不干燥的存在。 <ol><li>加几滴蒸压蒸馏水文化，以保持湿润（如文化干燥）。 </li><li>继续监察文化，并根据需要重复步骤1.7.1。 </li></ol></li><li>根据观察荧光显微镜流浪三龄幼虫第3节所述。 </li></ol> 2。感应浸润性肿瘤的<ol><li>使用表2中列出的股票为浸润性肿瘤的诱导。 </li><li>从步骤1.2以上的下列基因型使用起动机文化：Y，W，EY-FLP1;浴池- GAL80，FRT40A; Act5C&gt; Y +&gt; Gal4的，UAS-GFP </li><li>准备一个起始培养的“测试”股票下列基因型：W; LGL 4，FRT40A，UAS-V12的Ras / CYO </li><li>收集来自“测试仪”股票10只雌性童女和十男性从“测试”的股票。 </li><li>通过将他们两个在一个小瓶苍蝇F跨从“测试仪”和“测试”股票的男性和女性OOD。 </li><li>将交叉于25℃培养箱中培养，并让雄性和雌性交配和产卵24-48小时。 </li><li>检查小瓶足够卵沉积并为第一龄幼虫确保培养物不干燥的存在。 <ol><li>加几滴蒸压蒸馏水文化，以保持湿润（如文化干燥）。 </li><li>继续根据需要监控的培养干燥，然后重复步骤2.7.1。 </li></ol></li><li>在荧光显微镜观察三龄幼虫第3节所述。 </li></ol> 3。良性和浸润性肿瘤的观察<ol><li>使用三龄幼虫为良性肿瘤隔离和可视化。 <ol><li>湿油漆刷在蒸馏水中，并使用该漆刷从培养瓶的壁刮几三龄幼虫。 </li><li>将出现萧条的幻灯片用1X PBS幼虫，然后用湿油漆刷废料e关从幼虫表皮的任何食品原料。 </li><li>将幼虫放置在一个培养皿，并在-20℃下冷冻30分钟，离开固定的幼虫。 <ol><li> 另一种方法使用FLYNAP以固定幼虫。 </li><li>放置一个FLYNAP棒蘸FLYNAP到小瓶从3.1.3.2和堵塞小瓶让利插入小瓶幼虫后，静置30〜40分钟。 </li></ol></li><li>放置在玻片上固定化的幼虫，加一滴光卤烃油，盖上盖玻片，并根据与可视化绿色荧光蛋白（GFP）的能力，荧光显微镜观察。 </li><li>良性肿瘤轴承幼虫将发出绿色荧光的前部区域，其中头位络合物存在。 </li></ol></li><li>从文化上面的步骤＃2设置观察侵袭性肿瘤中选择10天的幼虫（诱导后）。第10天幼虫被选中是因为肿瘤侵入轴承幼虫有较长的升ARVAL阶段，通常不能pupariate。 </li><li>按照步骤3.1.1至3.1.4，除了代替三龄幼虫用10天的幼虫。 <ol><li>在第10天肿瘤侵入轴承幼虫将发出绿色荧光的前部区域，其中头位络合物存在。 </li></ol></li><li>如果在荧光显微镜配备有数码相机再取荧光良性和肿瘤侵入轴承幼虫的图片。 </li></ol> 4。良性和浸润性肿瘤的头侧复杂和进一步观察解剖幼虫的半透明表皮使得难以观察浸润肿瘤的程度。从而侵犯的程度更好地解剖头侧复杂了幼虫的可视化。应使用下列步骤来剖析头侧复杂。 <ol><li>用湿画笔选择三龄幼虫（良性肿瘤）和第10天的幼虫（浸润性肿瘤）FROM中的各培养瓶。 </li><li>幼虫放置在含冷的1X PBS解剖菜的好。使用油漆刷刮去从幼虫表皮的任何食物颗粒。 <ol><li>干净的幼虫转移到含有冷的1X PBS解剖盘的新鲜好。 </li><li>检查GFP荧光的使用能够检测荧光立体显微镜的存在。摒弃-GFP轴承幼虫。 </li></ol></li><li>加入冷的1X PBS的1.0毫升到一个新的良好的解剖盘，用一对镊子转移幼虫轴承无论是良性或浸润性肿瘤（解剖协议保持不变，这两种类型的肿瘤）。 </li><li>使用一对大约2/3rds的从前端钳握住幼虫下来。 <ol><li>使用其他钳子和巧妙的分隔幼虫的后三分之一个并丢弃。 </li><li>放手的幼虫的前2/3rds的。作为幼虫的内部压力被释放的内容幼虫会渗出体腔。 </li><li>用一对镊子除去肠，脂肪体和已渗出的体腔的幼虫的其它内容内。 </li><li>使用一对镊子举行幼虫的口钩将其推入幼虫表皮和使用其他钳子完全颠倒幼虫。 </li><li>使用镊子轻轻地，小心地取出脂肪体，唾液腺，肠道，翼盘复杂，任何气管导管。现在头侧复杂应该是可见的连接到倒置幼虫的口钩。大脑半球和腹神经索（VNC）仍然会通过神经从VNC发出连接到幼虫的表皮。 </li><li>使用镊子轻轻地打破头部神经和幼虫表皮之间的连接。 </li><li>由于VNC和幼虫表皮之间的神经连接断开和多余的脂肪体等组织取出，头侧合作mplex将变得清晰可见，并且将附着在表皮幼虫只在口钩。 </li><li>头侧复杂的可以留贴在幼虫表皮，如果复杂的需要固定的下游应用，如抗体染色或后可视化。 </li><li>如果没有进一步的下游应用会被执行，然后头侧络合物可从幼虫表皮被分离并置于滴基于安装介质进行进一步的观察和分析的甘油。使用VECTASHIELD作为安装介质。 </li></ol></li></ol>

<ol>
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C., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+Ras(V12)+and+scribbled+clones+induces+tumour+growth+and+invasion.">Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion.</a> Nature. 463, 545-548 (2010).</li></ol>

我什么都没有透露。

癌症是一种复杂的疾病有一个更好的了解，今天比过去。然而，仍有许多需要学习和解释之前，我们并不拥有相关机制的全貌。这里提出的简单的协议，能够诱导基因在整个生物体良性和侵入性肿瘤，然后研究与肿瘤的此模型中的进展有关的生物学。大部分在果蝇和其他生物的现有技术或者利用基于细胞的培养系统，以了解肿瘤发生及转移，或一个系统，它采用原发肿瘤组织移植入成年的?...

肿瘤从良性到转移状态的进展是明智步骤过程，其特征是存在于主体8的保护机制逃避。例如肿瘤细胞在体内必须能够逃避细胞凋亡和免疫系统，突破了专门的细胞外基质（ECM）称为基底膜，并克服了细胞周围的8所施加的社会控制。它是通过一步明智的进展，癌症细胞获得迁移和殖民遥远的地方在一个称为转移过程的能力。我们如何在肿瘤细胞克服了身体产生的障碍的认识仍处于起步阶段，但是，从研究新兴的画面迄今为止所做指向由癌细胞11-13重复使用正常的发育过程及信号通路。 果蝇果蝇得到了大幅我们UND贡献erstanding通过使用开发了在过去几十年中14-17先进的基因技术正常发育和疾病。使用我们已经到达了一个更好地了解各种癌基因和抑癌基因的18-22诱变和表达的工具。然而，肿瘤转移是一些已被主要研究了细胞培养模型23,24以及各种异种移植模型25-27的遗传病变之间的合作的结果。这些模型虽然厉害有其局限性，因为他们不完全模仿在活的有机体中发现的条件。此外，在现有的小鼠转基因模型是繁琐，不利于侵入行为28,29遗传分析。一些研究试图了解肿瘤细胞的侵袭果蝇 30,31。这些技术主要是利用原发肿瘤移植到主机，然后依靠跟踪TRAnsplanted肿瘤侵犯邻近组织32,33。一个强大的技术，称为MARCM 10适应了Pagliarini和徐肿瘤浸润在果蝇 9建模。肿瘤侵犯的这家优雅的遗传模型利用激活的癌基因和细胞极性的丧失之间的合作。这种造型的优势在于，所述浸润性肿瘤是在一个完整的有机体从而绕过需要组织移植产生的事实。带来的致癌合作，活化的癌基因类似的Ras V12是在细胞中表达的在幼虫眼触角光盘克隆。由于MARCM技术的结果，这些克隆还标有绿色荧光蛋白（GFP），方便的可视化和由合子细胞极性突变体像致命的巨型幼虫 ， 字迹潦草 ，和光盘大，其结果是在绿色荧光蛋白标记的浸润性肿瘤头侧复杂。在这个报告中，我演示如何诱导，并且无论是在一个完整的幼虫的上下文并在解剖出头侧复杂的可视化这些侵入性肿瘤。肿瘤诱导这里提出利用对果蝇第二染色体的试剂。在表2中，我提供股票上，可以被用于相同目的的X和第 3染色体的列表。我认为，这种简化的协议将使这种技术容易接触到感兴趣的研究人员了解肿瘤进展的分子基础。

<table border="1">
	<tbody>
 <tr>
 <td>10X PBS (phosphate buffered saline) pH 7.2 stock solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Chilled 1X PBS pH7.2 working solution</td>
 <td>Invitrogen, Sigma Aldrich</td>
 <td></td>
 <td>Make fresh and refrigerate, can be used up to a week</td>
 </tr>
 <tr>
 <td>Flynap</td>
 <td>Carolina Biologicals</td>
 <td></td>
 <td>Fly anesthesia needed to anesthetize larvae</td>
 </tr>
 <tr>
 <td>Fixative</td>
 <td>0.1M PIPES, pH 7.2, 4% Paraformaldehyde</td>
 <td></td>
 <td>Needed to fix the dissected cephalic complex</td>
 </tr>
 <tr>
 <td>Ice Bucket</td>
 <td>Several</td>
 <td></td>
 <td>Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube</td>
 </tr>
 <tr>
 <td>1.7ml Eppendorf tube</td>
 <td>Various</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Glass slides, cover glass</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield Mounting Media or any other mounting media</td>
 <td>Vector Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon 200 or 700 Oil</td>
 <td>Polysciences Inc. or Halocarbon.com</td>
 <td></td>
 <td>Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope</td>
 </tr>
 <tr>
 <td>Sally Hansen "Hard as Nails" nail polish</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td>Needed to seal the edges of Coverslip</td>
 </tr>
 <tr>
 <td>A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope</td>
 <td>Leica and others</td>
 <td></td>
 <td>Needed to observe the GFP fluorescence in larvae</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pyrex 9 well spot plate or any other dissection dish</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paint Brush</td>
 <td>Can be found at any general merchandise store</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Materials needed to perform the experimental protocol presented in this article.</td>
 </tr>
 </tbody>
 </table>

a protocol for genetic induction and visualization of benign and invasive tumors in cephalic complexes of drosophila melanogaster

果蝇照亮了我们的正常发育和疾病的遗传基础的了解，在过去的几十年，今天它仍然极大地促进我们对复杂疾病的1-7了解。肿瘤从良性到转移状态的进程是一个复杂的事件8，并已在模拟果蝇 ，以帮助我们更好地了解这种疾病9的遗传基础。在这里，我提出了一个简单的协议，以基因诱导，观察并分析肿瘤的果蝇幼虫的进展。肿瘤诱导的技术是基于MARCM系统10上，并利用活化的癌基因，的Ras V12和细胞极性基因丢失（ 乱写 ， 光盘大和致死巨型幼虫 ），以产生浸润性肿瘤9之间的合作。我将演示如何将这些肿瘤可在完整的幼虫进行可视化和那么如何将这些可解剖出作进一步分析。这里介绍的简化协议应该有可能使这种技术通过兴趣了解一个基因在肿瘤侵袭中的作用的调查予以确认。

作为协议的结果，在这里呈现的用户将能够通过过度激活的癌基因在幼虫眼触角成虫盘诱发良性肿瘤。用户也将能够通过过表达活化的癌基因在细胞的克隆以诱导侵入性肿瘤在眼触角光盘也突变体的细胞极性基因。肿瘤可以很容易地可视化，用荧光显微镜在整个幼虫或在已解剖出的幼虫体腔（ 图1）的头部复合物“绿色荧光”组织的帮助。良性肿瘤将被局限在眼睛触角盘复杂的GFP阳性...

Watch this Scientific Journal Video about A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster at JoVE.com

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

激活的致癌基因RAS一样的V12和细胞极性基因一样潦草 ，导致肿瘤生长的果蝇 ，其中肿瘤细胞也显示侵入行为的突变之间的合作。在这里，一个简单的协议为良性和浸润性肿瘤的诱导和观察提出。

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合<em&gt;果蝇</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

a-protocol-for-genetic-induction-visualization-benign-invasive-tumors

Research

JoVE Journal

Medicine

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

9.4K 次观看.  Western Kentucky University. 激活的致癌基因RAS一样的V12和细胞极性基因一样潦草 ，导致肿瘤生长的果蝇 ，其中肿瘤细胞也显示侵入行为的突变之间的合作。在这里，一个简单的协议为良性和浸润性肿瘤的诱导和观察提出。 

视频: A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

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 ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

小鼠横主动脉狭窄诱导左心室肥厚的技术研究

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

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 ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...