作者想感谢他们的技术援助，希高桥布赖恩Muntean，邵螺，和布莱尔梅尔。支持这项工作是由美国国立卫生研究院（DK080640）和托莱多和ProMedica转化研究激励奖苏里亚Nauli博士大学的奖励。

1。细胞预处理和中期准备<ol><li>杜尔贝科的修改鹰的5％CO 2培养箱中10-15％的胎牛血清（FBS），1％青霉素/链霉素在37°C间（DMEM培养基）中的细胞生长，直到他们达到70-80％汇合。 </li><li>治疗细胞秋水仙胺溶液与0.05微克/毫升30-60分钟。 </li><li>任何浮动细胞中含有50毫升无菌猎鹰离心管收集介质。板sterile1X-PBS漂洗细胞。后用无菌胰蛋白酶孵育1-2分钟，收获和收集到同一管内的剩余细胞。 </li><li> 1000 5分钟的转速旋转管，吸上清液留下0.5毫升，松开球，只用手指轻弹。 </li><li>根据颗粒大小，添加0.56％氯化钾DH 2低渗溶液5-10毫升，并在37°C孵育30-45分钟的暂停。 </li><li>添加一滴甲醇/醋酸（3:1​​体积/体积）每毫升低渗细胞悬液，轻轻颠倒混合管。 </li><li>在1200 rpm离心5分钟，收集沉淀步骤1.4，然后加入新鲜甲醇/醋酸（3:1体积/体积）固定液滴加5毫升，而轻弹颗粒continously。这个过程是关键，使中期利差不会被困在细胞的团块，这将危及实验。 </li><li>再次离心5分钟1200转，加5-10毫升的冰冷的甲醇/沿管壁醋酸固定液。在这个阶段，如果需要的话，这些细胞可以被保存在固定液收紧和短期或-80密封管在-20℃°C下长期（年）以供将来使用。 </li><li>无水乙醇清洁的幻灯片，然后浸在DH 2Ø为approximately10X的幻灯片，以形成滑动面水鞘。放置在玻璃板上和下降15-20μL细胞悬液的幻灯片（从1.8步放置在水浴在65-70°C间），10“以上的幻灯片。幻灯片为1-2分钟，晾干。 </li><li>干目标确保有中期染色体和利差是均匀分布的10X和40X光显微镜下检查的幻灯片。检查细胞质染色体周围存在。如果细胞质目前进行幻灯片预处理（胃蛋白酶消化），如果没有细胞质和染色体有良好的形态，那么就没有幻灯片预处理的需要。 </li></ol> 2。幻灯片预处理（胃蛋白酶处理） <ol><li>适用于120μL1:200酶溶液溶解到24毫米×60毫米显微镜盖玻片2X-SSC（20毫克/毫升）和中期反转滑面的玻璃罩上，然后轻轻地翻转中期滑面和孵育37°C为45分钟。 </li><li>小心地取出不刮伤幻灯片的玻璃罩和2X-SSC缓冲液洗在coplin壶每5分钟15分钟用颤抖的。 </li><li> 5-15μl蛋白酶库存解决方案的（DH 2 100毫克/毫升）加入到一个干净的烧杯中，然后加入100毫升预热（37℃）0.01M盐酸重要的是，胃被添加到一个干净的第一，不直接将盐酸溶液的烧杯中，否则它不会溶解在溶液中。孵育在coplin罐子含有盐酸/胃蛋白酶溶液，在37的幻灯片°C为3-5分钟。这一步是非常关键的太多消化会导致染色体overdigested太少消化细胞质将离开，未消化这可能会导致非特异性探针结合杂交信号的干扰。 </li><li>在coplin含5分钟，在室温下两次一罐100毫升1X-PBS洗幻灯片。 </li><li>洗的幻灯片，在coplin jar文件包含在室温下（1M MgCl 2的 50毫升，950毫升的1X-PBS）5分钟100毫升1X-PBS/MgCl 2。 </li><li>将在coplin罐子containin的幻灯片G 100毫升1％甲醛在室温（1X-PBS/MgCl 2 100毫升1.7毫升37％的甲醛）为10分钟。 </li><li>在coplin含5分钟一罐100毫升1X-PBS洗幻灯片。 </li><li>使用干40X镜头，以确保正常消化和幻灯片没有细胞质和染色体形态保留的光显微镜下观察的幻灯片。选择一个面积为杂交用金刚石笔。 </li></ol> 3。染色体和探针变性和杂交<ol><li>准备新鲜变性溶液（70％formamide/2X SSC，pH值7.0）和prewarm至70-80°C间，在coplin的jar包含在70水浴中变性的解决方案的coplin罐子放在水浴中放置在幻灯片°小鼠染色体和80 C度为30年代的1.5分钟的人类染色体。 </li><li>立即放置在冰冷的70％乙醇，80％的3分和100％的乙醇和空气干燥3分钟的幻灯片。检查幻灯片染色体的形态。好记黑暗染色体，而不是“相轻”或晕染色体染色体的形态。 </li><li>温暖的SkyPaint探头（天空油漆盒小瓶＃1）在37°C振荡20分钟，旋涡和离心机在1000 rpm短暂的几秒钟。 </li><li>在两个步骤的周期为5分钟周期由37℃60分钟允许为preannealing的标记的DNA探针在85°C编程1热循环变性探针。 </li><li> 10μL的变性探针应用到杂交和覆盖面积为22毫米×40毫米的玻璃罩确保不捕获的气泡。在37°C密封玻璃罩边缘用橡皮泥，在湿盒孵育48-72小时。 </li></ol> 4。荧光探针检测<ol><li>卸下玻璃罩仔细放置在一个coplin包含JAR的幻灯片预热（45℃）洗涤液（新鲜配制的50％2X SSC的甲酰胺）。洗5分钟45三次°C的振动水浴在45转</li><li>洗洗涤解决方案II（1X SSC）在45°C 5分钟两次用颤抖的幻灯片。 </li><li>解决方案三（4X SSC/0.1％吐温20）洗5分钟，45℃振荡Ç洗的幻灯片。 </li><li>阻断剂适用于80μL（天空油漆盒＃2小瓶），放置在37°C盖玻片孵育30分钟。 </li><li>删除幻灯片，让液体排出。适用于80μLCy5的染色试剂（浓缩抗体检测的CAD套件＃3小瓶）在37°C，适用于玻璃罩和孵育40分钟。 </li><li>洗洗涤溶液在45℃5分钟，用颤抖的第三三次幻灯片。 </li><li>适用于80μLCy5.5染色试剂（浓缩抗体检测的CAD套件＃4小瓶），放置在37°C玻璃罩和孵育40分钟。 </li><li>洗洗涤溶液在45℃5分钟，用颤抖的第三三次幻灯片。 </li><li>倾斜的幻灯片，并允许液体<html>流失。适用于20μL的DAPI的抗褪色试剂（天空油漆盒小瓶＃5），并放置一个24毫米×60毫米显微镜盖玻片。小心地取下空气泡沫可能已经形成。幻灯片可以立即成像，或保存于4°C，在不超过1周的黑暗。 </li></ol> 5。图像采集和分析<ol><li>通过查看中期幻灯片使用奥林巴斯显微镜配备了60X油浸没透镜，光谱立方体（定制设计的三带通滤波器），的DAPI过滤器与CCD相机Sagnac干涉仪模块，图像采集完成。 </li><li>光谱核型进行了使用后的天空视野软件（应用光谱成像版本1.62）的用户手册。 </li><li>染色体图像分析后，可以被看作（与特定的荧光颜色）的彩色图像，伪彩色图像（颜色分类）和倒置的DAPI图像（具体带型）。 </li></ol>e_title“&gt; 6代表结果。 一个完整的SKY过程通常需要一周左右的时间（ 图1）。这包括图像采集和分析提供中期细胞有充足的货源。染色体核型分析显示正常小鼠的染色体核型（40，XY）的野生型小鼠的细胞（ 图2a）。相比之下，PKD1细胞- / -小鼠（PKD的小鼠模型）显示在染色体数目和结构异常，如染色体缺失（8号染色体），易位（染色体11和19）（ 图2b），显着增加。我们还分析了从ADPKD患者的血管组织。一个简单计数染色体数目的研究表明，非多囊肾多囊肾血管样品，有23对染色体正常的数字（ 图2c）。然而，在一般情况下，我们观察到染色体分离失败，造成了46对染色体DPKD样本，而不是23对（ 图2d）。 <img alt="图1" src="/files/ftp_upload/3887/3887fig1.jpg" /> 图1。天空协议流程图。天空协议的流程图说明步骤来完成的实验，从细胞预处理和中期准备开始图像采集和分析。每一天一步一步的程序左侧大约一个星期的时间表。 <img alt="图2" src="/files/ftp_upload/3887/3887fig2.jpg" /> 图2。永生的小鼠细胞系和新鲜分离的人原代细胞的光谱核型分析。颜色和个别染色体倒置的DAPI图像显示之前的染色体排序。排序后，染色体是在“分类”表。提出了从野生型小鼠的中期蔓延）的天空图像包含染色体核型正常（40，XY）。 二） </st/ - -鼠标显示异常染色体数目（68 40）和染色体异常，如染色体缺失（8号染色体）和染色体易位（染色体11和19）ç）天荣&gt;从PKD1中期蔓延的天空图像从非多囊肾病人的血管组织的中期蔓延的形象，具有正常的核型（46，XX）。D）从多囊肾病人的血管组织的中期蔓延的天空图像包含的异常核型（92，XXXX的）。部分数据此前已报告和重用的许可14。

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没有利益冲突的声明。

光谱核型分析（天空）是一种细胞遗传学技术在研究基因和染色体组成。这种技术利用染色体涂染探针，这些探针的检测通过Sagnac干涉仪获得。完整的SKY过程通常需要大约一个星期，它涉及到几个关键步骤（ 图1）。天空采用了标准的协议，这是首次由帕迪拉纳什等 3。该协议已被适应各种细胞遗传学实验室，包括我们。 在协议中的关键步骤，质量...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td> cellgro敏达公司</td><td> 10-013-CV </td><td></td></tr><tr><td>胎牛血清（FBS）， </td><td>胎牛血清</td><td> SH30088-03 </td><td></td></tr><tr><td> penecillin /链霉素</td><td> Thermo Scientific的</td><td> SV30010 </td><td></td></tr><tr><td>秋水仙胺</td><td>罗氏诊断</td><td> 10 295 892 001 </td><td> 10微克/毫升</td></tr><tr><td>盐酸</td><td> Fisher Scientific则</td><td> A144-500 </td><td></td></tr><tr><td>氯化钾</td><td> Fisher Scientific则</td><td> S77375-1 </td><td></td></tr><tr><td>磷酸盐缓冲液</td><td> Thermo Scientific的</td><td> SH30256-01 </td><td></td></tr> &lt;TR&gt; <td>天空油漆探头套件（人力） </td><td>应用光谱成像</td><td> SKY000028 </td><td></td></tr><tr><td>天空油漆探头套件（鼠标） </td><td>应用光谱成像</td><td> SKY000030 </td><td></td></tr><tr><td>浓缩抗体检测试剂盒</td><td>应用光谱成像</td><td> SKY000033 </td><td></td></tr><tr><td>胰蛋白酶</td><td> Thermo Scientific的</td><td> SH30236.01 </td><td></td></tr><tr><td>甲醇</td><td> Fisher Scientific则</td><td> A433P-4 </td><td></td></tr><tr><td>醋酸</td><td> Fisher Scientific则</td><td> A38-212 </td><td></td></tr><tr><td> RNA酶A </td><td>罗氏诊断</td><td> 10 109 169 001 </td><td></td></tr><tr><td>胃蛋白酶</td><td> Sigma-Aldrich公司</td><td> P6887-5G </td><td></td></tr><tr><td> MgCl 2的 </td><td> Fluka公司</td><td> 63069-500ML </td><td></td></tr><tr><td> 37％的甲醛</td><td> mallinckrodt贝克</td><td> 2106-02 </td><td></td></tr><tr><td> 20X SSC的</td><td> Promega公司</td><td> V4261 </td><td></td></tr><tr><td>甲酰胺</td><td> Fluka公司</td><td> 47671 </td><td>准备只是在使用前</td></tr><tr><td>吐温20 </td><td> Fisher Scientific则</td><td> BP337-500 </td><td></td></tr><tr><td>显微镜的玻璃幻灯片</td><td> Fisher Scientific则</td><td> 12-549 </td><td></td></tr><tr><td>显微镜盖玻璃24x60mm </td><td>厂商VWR Vista的视觉</td><td> 16004-312 </td><td></td></tr><tr><td>橡皮泥</td><td>埃尔默（当地的零售商店） </td><td></td><td></td></tr><tr><td>杂交/ humidifiedchamber的/托盘</td><td> simport </td><td> M920-2 </td><td>在底部放湿纸巾</td></tr><tr><td>热基因扩增仪</td><td> Eppendorf公司</td><td> epgradient小号</td><td></td></tr><tr><td>振动平台/轨道摇床</td><td> bellco生物技术</td><td></td><td></td></tr><tr><td>振动/水浴</td><td>精密科学</td><td></td><td></td></tr><tr><td>的DAPI过滤立方体</td><td>色度技术</td><td></td><td></td></tr><tr><td>天空过滤立方体</td><td>色度技术</td><td></td><td></td></tr><tr><td> SpectraCube </td><td>应用光谱成像</td><td></td><td></td></tr><tr><td>倒置细胞培养显微镜</td><td>尼康</td><td>尼康的Eclipse TS100 </td><td></td></tr><tr><td>荧光显微镜</td><td>奥林巴斯</td><td> IX70 </td><td> 60X油</td></tr></tbody></table>

spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease

常规的方法来识别和分类 ​​，个别染色体取决于每个染色体的模式，在一个特定的物种正在分析1，2独特的带。然而，这种古典的带技术，是不可靠的识别复杂的，如那些与癌症相关的染色体畸变。为了克服带技术的局限性，光谱核型分析（SKY）介绍，提供许多染色体异常可靠的信息。 天空是多色荧光原位杂交（FISH）技术检测与中期染色体光谱显微镜3，4。天空已被证明是一个有价值的工具为一个范围广泛，大量的遗传性疾病和恶性肿瘤的5，6相关的染色体异常的细胞遗传学分析。天空涉及使用多色荧光标记的DNA探针准备从退化寡通过PCR otide引物。因此，每一个染色体有独特的光谱颜色后原位杂交探针，这些差异标记了荧光染料的混合物（罗丹明，得克萨斯红，Cy5标记，FITC标记和Cy5.5）。漫天的探头由55号染色体特异性探针7-10。 漫天的过程涉及几个步骤（ 图1）。天空要求高的有丝分裂指数从正常或病变的组织或血液细胞的可用性。从一个新鲜分离的原代细胞或细胞系单细胞的染色体玻片上传播。不同组合的每个染色体的特定荧光染料标记染色体蔓延。探头检测和图像采集，光谱成像系统由Sagnac干涉仪和CCD相机。这使得从样品发出的可见光光谱测量，获得光谱图像来回米个人染色体。 HiSKY，软件，用来拍摄的图像分析结果提供了一个易于识别的染色体异常。最终的结果是一个中期和核型分类的图像，每对染色体中有不同的颜色（ 图2）。这使得容易识别染色体的身份和易位。有关详细信息，请访问应用光谱成像网站（ <a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a> ）。 天空最近被用于鉴定染色体分离的缺陷和染色体异常在人类和小鼠的常染色体显性多囊肾病（多囊肾），一种遗传性疾病，特点是在初级纤毛功能障碍11-13。使用这种技术，我们证明了染色体分离异常和染色体缺陷的存在，在多囊肾患者和小鼠模型14。进一步分析使用的SKY不仅让我们能够找出染色体数目和身份，但也准确地检测出非常复杂的染色体畸变，如染色体缺失和易位（ 图2）。

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

光谱核型分析（天空）是一种先进的细胞遗传学技术，以确定基因和染色体畸变。这种技术利用染色体涂染探针，这让所有染色体的分类。天空也可以识别复杂的染色体畸变，在小鼠和人类的各种疾病，包括多囊肾和种族隔离的缺陷。

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Research

JoVE Journal

Medicine

38.2K 次观看.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. 光谱核型分析（天空）是一种先进的细胞遗传学技术，以确定基因和染色体畸变。这种技术利用染色体涂染探针，这让所有染色体的分类。天空也可以识别复杂的染色体畸变，在小鼠和人类的各种疾病，包括多囊肾和种族隔离的缺陷。

视频: Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

科研

医学

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 ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

对称Bihemispheric死后的大脑切割学习健康和病理条件大脑在人

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

在几种肾脏疾病小鼠模型中使用聚乙烯亚胺纳米颗粒将外源人工合成的miRNA模拟物递送到肾脏

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...