We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

在这项研究中使用的所有动物根据ARVO声明动物在眼科和视觉研究中使用的照顾和处理。 1.生产重组腺相关病毒（腺相关病毒）由HEK-293细胞17,18的瞬时转染的注：请参阅麦克卢尔C，朱庇特（2011）19。 <ol><li>纯化大规模质粒制备的AAV载体质粒（至少1毫克/毫升）。使用3质粒。所述AAV辅助质粒携带复制和衣壳基因 ​​未经AAV反向末端重复的ITR，所述转基因表达盒携带AAV ITR称为转移质粒，并且该质粒供给腺病毒辅助基因的E2，E4和VA RNA基因所指为pHELPER。 </li><li>转染293细胞使用聚乙烯亚胺（或其他转染试剂）这3个质粒。高于80％的转染效率是必要的良好的六RAL产量。 </li><li>净化从293细胞裂解液重组AAV上的碘克沙醇梯度。 15％，25％，40％，和60％的碘克沙醇被用于获得梯度。收集含AAV颗粒的40％的碘克沙醇馏分，浓缩超速离心后的级分在500000×g离心对PBS与0.001％的Pluronic 1小时，缓冲液交换（磷酸盐缓冲盐水）。 </li><li>要确定病毒基因组滴度20，进行DNA酶蛋白酶K消化后QPCR。正向和反向引物扩增位于AAV2 ITR的62 bp的区域。对于质粒的标准，该探针用FAM和具有黑孔猝灭剂（BHQ）。 </li><li>进行定量PCR（定量聚合酶链式反应）中的25微升用340纳米的终体积反向ITR底漆，100nM的正向引物的ITR，100nM的AAV2 ITR探针，0.4毫的dNTPs，2mM的氯化镁，1个白金Taq缓冲液，1U白金Taq和5微升模板（标准，样品和无模板对照）。 </li><li>为的标准中，使用的质粒转染在7×10倍连续稀释液（每2×10 9个至2×10 3产生的最终浓度从10月10日至4月 10日的基因组/毫升的基因组/毫升5微升）。 </li><li>由初始变性步骤在95℃下进行15分钟，随后变性的40个循环的开始的程序，在95℃，1分钟退火/延伸60℃，1分钟。储存在4℃下短期储存，或在-20℃下长期储存。 </li></ol> 2.玻璃体内注射AAV的<ol><li>麻醉C57BL6J小鼠用氯胺酮（50毫克/千克）和赛拉嗪（10毫克/公斤），并验证翻正反射时，撤回反射和尾部夹送响应指示一个适当的麻醉的损失。由neosynephrine 5％的角膜应用扩张的学生和mydriaticum 0.5％眼药水。使用上的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>通过超细地下30 disposab乐针穿过赤道旁缘，入玻璃体腔（见邱K，朱庇特（2007）21）。注入1微升原料含有1至4×10 11 VP ShH10-GFP或AAV5-GFP与直接观察在玻璃体腔的中心针（ 图1）。用一只眼睛作为ILM实验对照。注入两只眼睛的BRB实验。对注入的眼睛敷上消炎和抗菌局部治疗。 </li><li>扩张与学生和neosynephrine mydriaticum眼药水成像。执行眼底检查与眼底照相机跟随的GFP（绿色荧光蛋白）的表达1周，2周，1个月，和玻璃体内注射后的2个月（ 图1）。用CO 2吸入1〜2个月后喷射牺牲小鼠。 </li></ol> 3.免疫组化<ol><li>视网膜的冷冻切片（ 图1），解剖剜出眼睛去除升在4％多聚甲醛1小时ENS和角膜，并浸泡修复。 <ol><li> Cryoprotect先前固定的眼睛在10％蔗糖1小时，在室温下，20％的蔗糖为另一小时，在室温下进行。 Cryoprotect在30％蔗糖溶液，过夜，在4℃。冻结和嵌入树脂如低温矩阵嵌入眼罩。使10微米的冰冻切片和安装在滑轨经过特殊处理的冷冻组织切片染色以及在改善组织粘连。 </li><li>透用0.1％的Triton X100的部分在PBS pH 7.4的5分钟。洗涤2次在PBS中，块1小时，在室温下在PBS中以1％BSA，0.1％吐温20。 </li></ol></li><li>视网膜flatmounts（ 图1），固定摘除眼睛用4％多聚甲醛中15分钟，以方便清扫。 <ol><li>在15-30分钟，解剖眼睛去除角膜和晶状体。通过切割周围锯齿缘分离的视网膜色素上皮细胞（RPE）和巩膜视网膜和视神经。沉浸在4％多聚甲醛另外30分钟来固定视网膜和在转导视网膜细胞的荧光蛋白（固定必须不超过24小时）。在无菌PBS，pH 7.4中洗涤5分钟。 </li><li>改变的PBS以封闭缓冲液（PBS中的1％BSA，2％正常山羊或驴血清，0.5％的Triton X-100），并孵育在封闭缓冲液为任一4小时，在室温下或4℃过夜。 </li></ol></li><li>孵育组织与初级抗体在封闭缓冲液为任一4小时，在室温下，或在4℃下过夜。洗涤3次，用PBS。 注意：是使用的抗体如下：抗层粘连蛋白（1 / 1,000）标记在ILM，抗视紫红质克隆4D2（1/500）标签棒，防谷氨酰胺合成克隆GS-6（1/1500）标签Müller细胞，PNA凝集素（1/40）标签锥。 </li><li>孵育组织用1：500稀释的Alexa Fluor缀合的第二抗体在封闭缓冲液中进行1小时（冷冻切片）或2小时（flatmounts），在室温坦佩叉涂抹并避光。洗涤3次，用PBS。 </li><li>使减轻削减视网膜和flatmount它与上无论是感光或视网膜神经节细胞（RGC）侧朝上载玻片。添加水mountingmedium，应用盖玻片并储存在4℃。 </li><li>执行激光扫描共聚焦显微镜共聚焦显微镜。由线获取图像顺序地，线，以减少激发和发射串扰。根据采样定理确定步长大小。使用曝光设置，最大限度地减少过饱和像素的最终图像。处理的12位图像斐济，项目的Z部分上使用下的Z-项目函数的最大强度并最终转换为8位的RGB色彩模式的单一平面。 </li></ol>小鼠血液样本4. PCR分析<ol><li>注入100μl的原料含有的AAV-GFP的1至4×10 11颗粒进入麻醉C57BL6J小鼠的阴茎静脉（异氟醚的5％用于使用胰岛素注射器用超细6毫米针诱导密切室，并通过在手术过程中一鼻锥异氟烷2.5％）。这种动物将作为循环的AAV颗粒的阳性对照。 </li><li>从鼠尾注射和3小时，24小时，2天，以及之后ShH10玻璃体内注射或intrapenile喷射（ 图1）后第3天前样品的血液。约10-20微升被收集在肝素管。在手术完成之后，牺牲小鼠通过CO 2吸入。 </li><li>提取使用你选择的提取试剂盒血液样本的基因组DNA。 </li><li>执行的基因组DNA的PCR扩增。此协议使用下列引物对：绿色荧光蛋白，有义：5&#39;-CGACACAATCTGCCCTTTCG-3&#39;，反义5&#39;-CATGGACGAGCTGTACAAGGGA-3&#39;。以下PCR程序进行：95℃2分钟（1个循环）; 95℃45秒，55℃1分钟，72℃45秒（30个循环）; 72°C 5分钟（1周期）;4℃保持。 </li></ol> 5.可选埃文斯蓝方法注：通过测量血管白蛋白漏入使用伊文思蓝法10视网膜量化血管通透性。 <ol><li>通过溶解在通过5μm过滤器的生理盐水溶液（6毫克/毫升），超声处理5分钟，在超声波洗涤机，并过滤制备伊文思蓝染料。 </li><li>麻醉C57BL6J小鼠（异氟烷吸入，见步骤4.1），检查翻正反射，撤退反射和尾掐响应，表示适当的麻醉的损失，并通过阴茎静脉（ 图1），注入伊文思蓝（45毫克/千克）。检查爪子，枪口，耳朵小鼠变得清楚蓝色以下注射Evans蓝染料，证实了染料的吸收和分布。 </li><li>注射用氯胺酮（50毫克/千克）和赛拉嗪（10毫克/千克）的染料后麻醉C57BL6J小鼠3小时，并验证rightin的损失克反射表明适当麻醉。样品心内约500微升的血液在肝素管，并通过左心室用柠檬酸盐缓冲液灌注小鼠，持续2分钟（0.05M，pH为3.5;溶解7.8克柠檬酸和3.8克柠檬酸钠在1升的蒸馏水中）预热至37℃，以防止血管收缩。小鼠经由灌注处死。 </li><li>后灌注，剜除，仔细解剖手术显微镜，收集视网膜下两只眼睛（见第3.2节）。在离心蒸发器中干燥视网膜过夜，称重，并通过培养视网膜用100μl甲酰胺中18小时，在65℃振荡（100 XG）提取伊文思蓝染料。在30K的ω过滤管在20,798.5 xg离心2小时，在4℃离心机视网膜样品离心血样在20,798.5 xg离心15分钟，在4℃。 </li><li>使用这两种上清测量吸光度。减去最大吸光度FO测定各样品的吸光度řEvans蓝在620nm到最小吸光度在740纳米。从伊文思蓝中甲酰胺的标准曲线计算出的伊文思蓝浓度在血浆和视网膜。表达BRB渗透性在每小时每克干视网膜（微升血浆/克视网膜干重/小时）的伊文思蓝的微升。 </li></ol> 6.可选荧光血管造影注：泄漏从视网膜血管小鼠可通过腹膜内注射荧光素钠被可视化。 <ol><li>麻醉C57BL6J小鼠（异氟烷吸入，见步骤4.1）和瞳孔扩张与眼药水（neosynephrine和mydriaticum）。使用上的眼睛兽医药膏，以防止干燥，而在麻醉下。 </li><li>注入腹膜内0.01-0.02毫升10％荧光素钠在0.1ml无菌盐水进行荧光血管造影。 </li><li>收集双眼用眼底照相机注射后的图像。后亲的CO牺牲小鼠吸入2cedure。 注：20秒所必需的视网膜血管，以成为对造影可见。图片必须要3-10分钟最大荧光素注射后抓获。漏点（如果存在）2.5分钟后可观察到。在稍后的时间点的图像质量由于荧光素钠扩散到用于分析之后不久得到的玻璃体，因此仅图像丢失。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+adeno-associated+virus-mediated+gene+delivery+in+retinal+degeneration.">Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.</a> Hum Gene Ther. 21 (5), 571-578 (2010).</li><li>Sene, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+implication+of+Dp71+in+osmoregulation+and+vascular+permeability+of+the+retina.">Functional implication of Dp71 in osmoregulation and vascular permeability of the retina.</a> PLoS One. 4 (10), e7329 (2009).</li><li>Benard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Quantifiable+Blood+Retinal+Barrier+Breakdown+Model+In+Mice.">A New Quantifiable Blood Retinal Barrier Breakdown Model In Mice.</a> ARVO Annual Meeting. , (2011).</li></ol>

The authors have nothing to disclose.

该BRB调节血液和视网膜之间的分子交换。其击穿与各种疾病，如糖尿病性视网膜病或年龄相关性黄斑变性（AMD）相关联。我们最近发现，在一个肌营养不良蛋白敲除小鼠，其中显示可渗透BRB，视网膜变得更宽容的由腺相关病毒载体（AAV）介导的基因传递。然而，尽管BRB渗透性的AAV颗粒喷入眼内保持局限于此模型中的眼隔室。我们的研究结果表明，基因治疗中显示渗透BRB并不代表全身副作用附加险?...

Müller细胞是视网膜的主要神经胶质成分。形态，它们跨越视网膜径向和它们endfeet，与玻璃体接触，面对在ILM和后者的秘密分量。在ILM是一种基底膜约十个不同的细胞外基质蛋白（层粘连蛋白，聚集蛋白，串珠素，巢蛋白，胶原蛋白和一些硫酸肝素蛋白聚糖）组成。在开发过程中，它的存在是不可或缺的视网膜组织发生神经节细胞1-3，视神经轴突的导航和生存。但是，ILM是非本质的成人视网膜，可以在某些病理手术切除，而不会造成视网膜损伤4。在基因治疗中，此膜变成为使用自动增值服务的视网膜高效转导通过玻璃体内注射5的物理屏障。 通过他们的流程的广泛树枝状，Müller细胞提供营养和监管suppoRT既视网膜神经细胞和血管细胞。 Müller细胞也参与视网膜动态平衡的调节，在BRB 6的形成和维持。紧密连接视网膜毛细血管内皮细胞之间，Müller细胞，星形胶质细胞和周形成BRB。 BRB防止某些物质进入retina.In许多疾病，如糖 ​​尿病性视网膜病变，视网膜静脉阻塞和呼吸系统疾病，视网膜缺氧会导致通过BRB 7-9泄漏。这种断裂是与增加血管通透性，导致血管性水肿，视网膜脱离和视网膜损伤相关联。 Müller细胞被紧密地与血管和基底膜相关联，打在两BRB和ILM完整性中起重要作用。因此，标记缪勒胶质细胞是特别相关的这些视网膜屏障的物理状态的研究。 经典同盟，BRB渗透性是用伊文思蓝测定法，包括全身性注射Evans蓝染料，其结合非共价地与血浆白蛋白的测定。此法测量从血管进入视网膜（见协议第5条）10白蛋白漏出（蛋白质中等大小，〜66 kDa的）。另外，血管渗漏可以通过荧光血管造影视网膜的证明荧光素渗漏进行可视化（小分子〜359达;见协议第6条）11。然而，这两种方法允许BRB渗透性小分子和蛋白质的评估，但它们不提供关于在ILM的完整性信息。 因此，为了研究BRB渗透性，我们使用的AAV基础的方法，让上BRB渗透性至较大的分子（ 例如，AAV颗粒，25纳米的直径）的信息。事实上，我们的方法可以检测出的AAV转基因的存在于血液中，这将表明，〜25 nm的直径的颗粒会能够渗透到血液中。这种方法还提供了在病理条件下在ILM和细胞外基质蛋白质的结构信息。两个变种AAV对于这样的研究有用：AAV5和ShH10。视网膜下注射，AAV5具有天然向性为感光细胞和视网膜色素上皮细胞12，但是它不能获得跨越到外视网膜给药时进入玻璃体在野生型视网膜中有完整的ILM 5,13。 ShH10是已工程化以特异性靶向神经胶质细胞在神经元14,15的AAV变种。 ShH10选择性标记在健康和患病的视网膜与视网膜增加的效率受损障碍16Müller细胞。再加immuhistochemistry和血液DNA分析这些病毒的工具提供的视网膜屏障的状态和它们在疾病的参与（ 图1）的信息。

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using adeno-associated virus as a tool to study retinal barriers in disease

Müller细胞是视网膜的主要神经胶质细胞。其最终的脚形成的视网膜在外侧和内界膜（ILM）的范围内，并在与星形胶质细胞，周细胞和内皮细胞结合它们建立血 - 视网膜屏障（BRB）。 BRB限制了血液和视网膜之间的物质运输而在ILM充当基底膜限定组织学视网膜和玻璃体腔之间的边界。标记Müller细胞是特别相关，研究视网膜屏障的物理状态，因为这些细胞是BRB和ILM的一个组成部分。既BRB和ILM经常改变视网膜病和负责疾病症状。 有几种公认的方法来研究的BRB的完整性，如Evans蓝测定法或荧光血管造影。然而，这些方法不提供关于BRB渗透性吨的范围内的信息Ø较大的分子，在纳米范围内。而且，它们不提供对其他视网膜屏障如在ILM的状态信息。研究BRB渗透性沿着视网膜ILM，我们使用的AAV基础的方法，可提供对渗透性BRB对较大分子虽然表示在疾病状态在ILM和胞外基质蛋白的状态的信息。两个变种AAV对于这样的研究有用：AAV5和ShH10。 AAV5具有天然趋光感受器，但它不能得到跨越到外视网膜给药时进入玻璃体时在ILM是完整的（ 即，在野生型视网膜）。 ShH10具有朝向胶质细胞强烈的取向和在健康和患病的视网膜将选择性标记米勒胶质细胞。 ShH10提供视网膜更高效的基因传递，其中ILM受到损害。再加上免疫和血液DNA分析这些病毒工具揭示到的视网膜疾病障碍的状态。

我们预计增加的使用ShH10缪勒胶质细胞的视网膜转导，如果该动物模型显示扰动在ILM（ 图2A - B）的结构。例如，我们已经表明，在不存在的Dp71，ShH10靶特异性而更有效地缪勒胶质细胞通过玻璃体内注射，这表明在ILM的通透性增加在该鼠标线相比野生型小鼠16（ 图2C - F）。 AAV5也可以用作ILM渗透性的指标。 AAV5，无效通...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

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14.9K 次观看.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

视频: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

科研

医学

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

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

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 ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

整骨手法治疗为肺炎是非常好的辅助工具

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

眼底摄影作为一种方便的工具，研究微血管回应流行病学研究心血管疾病的风险因素

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

适应人类Videofluoroscopic燕子研究的方法来检测和检定吞咽困难的小鼠疾病模型

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

利用视网膜成像研究痴呆

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

消化不良教育工具作为消化不良管理中的一种新帮助

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

小动物模型中腺相关病毒载体的结膜下给药

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...