这项工作是支持部分由美国耳科到CAM协会的赠款和大学医院案例医学中心KNA和CAM的赠款。我们要感谢杰夫Churney聚焦医药集团有限公司（辛克利，俄亥俄州）为我们提供用显微镜摄像头。提供融合创意营销公司（瓦尔多斯塔，GA）提供视频制作服务，我们感谢他们的意见，在编制的视频在凯斯西储大学的乔纳森Frankel和亚历莱维特。

第1部分：麻醉和手术的准备<ol><li>成年后，查尔斯河国际实验室公司（威尔明顿，马萨诸塞州）的女性邓肯 - 哈特利几内亚猪是氯氨酮60毫克/公斤和甲苯噻嗪12毫克/公斤肌肉注射麻醉。 </li><li>石油润滑油适用于眼睛，以防止由于干燥，角膜损伤的动物。 </li><li>动物是从头顶到肩膀和耳朵之间剃光。 </li><li>剃光的面积是prepped先用7.5％碘伏70％的乙醇在无菌时尚。 </li><li>动物被放置到一个与气候变暖毯的手术台，到了一个平台，提升头部和颈部弯曲。动物的头是远离外科医生。颈部有足够的屈曲是必需的，以便充分暴露枕骨嵴。 </li><li>该动物是在无菌的时尚披。 </li></ol>第2部分：切口，暴露骨枕<ol><li> 0.25％布比卡因加肾上腺素（Marcaine）是沿准切口部位（枕部沿背中线切开皮肤）。这将导致局部血管收缩，限制出血，和提供持久的手术后镇痛的动物苏醒后。 </li><li>中线背侧皮肤切口是沿枕部头皮上。 <img src="/files/ftp_upload/1728/1728fig1.jpg" alt="图1" /> 图1：皮肤切口是沿背中线，如上图所示。 </li><li>为了获得足够的曝光，皮肤回缩横向切口两侧放置在一个小弯止血钳。 </li><li>一个横向切口，沿颈部肌肉，暴露骨枕。在这个阶段，由于枕大神经切断，尽管有足够的麻醉的动物可能会抽搐。脊髓横断横向切口进入枕下地区的危险。确保底层骨总是觉得下面的手术刀刀片，可避免这种并发症。 </li><li>枕下肌肉切骨，第三止血用于收回优越的肌肉边缘。止血是手术助理举行。 </li><li>一个Lempert骨膜电梯是用来揭露颞枕部缝合。 </li></ol>第3部分：钻井颞枕部缝合<ol><li>颞枕部缝合（图2），耳科钻一个1.5毫米的钻石毛刺曝光后用于钻inferio横向缝合。 </li><li>下面的头骨外部表，海绵质地的板障（松质骨的头骨的表之间的干预）将变得可见。一旦钻了板障，于颅骨内表出现，伴随着淡淡的蓝色色调，乙状窦。 </li><li>乙状窦的边缘定义无须穿透容器。 </li><li>一旦得到了充分的薄覆乙状窦骨，利用更高的放大倍率和直接用于小心取出覆乙状窦骨。 </li></ol><img src="/files/ftp_upload/1728/1728fig2.jpg" alt="图2" /> 图2钻部分的面积覆盖在颞枕部的缝合。 第4部分：管道阻塞<ol><li>中间镂空的乙状窦缩回来揭示的鳃盖。 </li><li>一个0.5毫米的钻石毛刺是用来钻的鳃盖内侧，进入内淋巴囊和管。 </li><li>淋巴管，然后使用直接与骨蜡包装。 </li><li>颅骨缺损是加强与明胶海绵（辉瑞公司，纽约，纽约）。 </li><li>使用4-0 POLYSORB（美国外科，诺瓦克，CT）缝合在一起带来的肌肉。有那么一个无缝地过渡到一个关闭皮肤切口皮下缝合技术。有了这个封闭有没有必要去除缝合之后，是没有风险的，动物爪在现场拉出一期缝合。 </li></ol>第5部分：手术后护理豚鼠被置于一个孵化器，以保持体温，并在30分钟的时间间隔检查，直至清醒，并能保持胸骨位置。动物是检查呼吸速率和车身颜色。该中心使用恩诺沙星（22.7毫克/毫升，皮下注射0.2毫升）和丁丙诺啡（0.3mg/mL，皮下注射0.05毫升），手术后感染的预防和控制疼痛。一次手术当天，两次手术后一天一个和两个的止痛药。虽然抗生素是没有必要的无菌苏rgery，为期五天，两次预防性应用抗生素的日子当然可能是手术后的第三天givenonce。 并发症及处理<ol><li>呼吸抑制，外科医生应始终了解动物的小号呼吸。如果呼吸减慢或停止，停止手术，目视检查胸运动。如果动物出现呼吸困难或呼吸困难，检查口和呼吸道不闭塞。 </li><li>乙状窦渗透使用明胶海绵和压缩吸尖，直到出血已停止。 </li><li>硬脑膜渗透与明胶海绵覆盖的缺陷。 </li><li>前庭损害不要超过1毫米，横向到乙状窦钻。这样做的风险打伤后半规管，这将豚鼠的所有数据无效。 </li></ol>

<ol>
	<li>Andrews, J. C., Bohmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surgical+approach+to+the+endolymphatic+sac+and+the+cochlear+aqueduct+in+the+guinea+pig.">The surgical approach to the endolymphatic sac and the cochlear aqueduct in the guinea pig.</a> Am J Otolaryngol. 10, 61-66 (1989).</li><li>Kimura, R., Schuknecht, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranous+hydrops+in+the+inner+ear+of+the+guinea+pig+after+obliteration+of+the+endolymphatic+sac.">Membranous hydrops in the inner ear of the guinea pig after obliteration of the endolymphatic sac.</a> Pract Otolaryngol. , (1965).</li></ol>

动物实验机构的动物护理和使用凯斯西保留地大学委员会规定的准则和法规的规定执行。

在手术阻塞淋巴管下面的几个星期，动物受经验波动感音神经性听力损失的，由听觉诱发脑干反应测量。听觉脑干反应（ABR）记录样品如图3所示。左耳作为unoperated控制。 <a href="https://www.jove.com/files/ftp_upload/1728/1728fig4.jpg"><img border="0" src="/files/ftp_upload/1728/1728fig3tmb.jpg" alt="图3" /></a> 图3：从豚鼠手术28周后，在16千赫的ABR阈值水平。左（unoperated）耳的阈值是30 dB声压级和权（经...

surgical induction of endolymphatic hydrops by obliteration of the endolymphatic duct

手术诱导的内淋巴积水豚鼠（ELH）闭塞和淋巴管阻塞是一种广为接受的条件和人类美尼尔氏病的一个重要的关联的动物模型。 1965年，罗伯特木村和哈罗德Schuknecht首次描述为淋巴管阻塞硬膜的方法（木村1965年）。虽然有效，这种技术，这需要对大脑的保护层的渗透，在受试动物的发病率和死亡率产生不良的水平。因此，安德鲁斯和Bohmer开发硬膜外方法，可预见的产生与中枢神经系统（CNS）的渗透。（1989年Andrews和Bohmer）相关的并发症少这里描述的硬膜外方法，首先需要在枕部，露出底层肌层地区正中切口。我们只在右侧。覆组织的适当回缩后，被制作成一个横向切口右枕部肌肉暴露的右颞枕部的缝合线。骨立即inferio外侧缝合线（图1），然后与耳科钻钻，直到乙状窦变得可见。乙状窦内侧回缩揭示了淋巴管，里面有内淋巴囊鳃盖。钻内侧的鳃盖成的区域内淋巴囊揭示了淋巴管，然后用骨蜡产生阻塞，并最终ELH包装。 在接下来的几个星期中，动物会表现出进步，波动性听力损失和ELH组织学证据。

Watch this Scientific Journal Video about Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct at JoVE.com

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

本视频演示了如何手术阻挠豚鼠内淋巴管内淋巴积水。

内淋巴管闭塞手术诱导的内淋巴积水

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

surgical-induction-endolymphatic-hydrops-obliteration-endolymphatic

Research

JoVE Journal

Biology

11.3K 次观看.  Case Western Reserve University. 本视频演示了如何手术阻挠豚鼠内淋巴管内淋巴积水。

视频: Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

成像效应在耳朵上的记忆体T细胞诱导后收养的DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

科研

生物学

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion.  Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction.  Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm.  This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.

Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement.

显微剪辑闭塞大脑中动脉瘤术中流量评估

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...