RIKEN - BSI我们感谢我们的同事为他们的好心的支持和富有成效的意见，在此工作。我想滋森教授，博士，教授摹BOR灶火SZ表示衷心的感谢。 Kiyoji松山，卡塔琳AK缂丝和RP ð Dobolyi谁劝我在日本高级研究的研究生，研究生课程学习，微透析方法。最后，我们感谢克里斯先生Sarnecky Akitsugu高须幸雄博士在其支持视频录制。

第1步。制备探针植入<ol><li>麻醉（在视频中未显示） 麻醉interperitoneal一个氯胺酮注射液（12.5毫克/公斤体重）和甲苯噻嗪（25毫克/公斤）的混合物，手术麻醉产生一个稳定的水平每只鸟。 </li><li>修正了一个头，一个立体框架 使用Xylocaine（TM）果冻耳棒“的压力，以减少当地的阵痛 。 鸟头是固定在耳酒吧和喙的持有人，持有在45度角的头组成的立体设备。 在立体定向手术植入，手术垫热控温暖的鸟身（温度= 34〜36℃）。 </li><li>消毒手术区 isodine（TM）手术前消毒术野。 </li></ol>第2步。立体定向定位<ol><li>参考点对齐 将虚拟探头（玻璃微管）的Lambda（Y点）和标记的Y点与染料填充吸管。其余葡 ​​聚糖TMR最后示踪研究是在视频中使用 。 </li><li>切口的皮肤 清除皮肤，暴露颅骨表面。然后，清洁用棉签头骨表面。 </li><li>虚拟导管定位（水平方向） 移动虚拟导管到目标站点（AP轴和横向协调） </li><li>小开颅手术和切开硬脑膜。 用于开颅罚款钳和罚款的注射针（30克）的硬脑膜切口 。 然后，干净的头骨表面。 </li><li>虚拟导管最终定位（植入） 在0.30毫米以上的微透析探针的预定目标区域定位引导导管的尖端 。 最后，再次清洁头骨表面，因为表面的清洁和干燥是艰难的粘接要求。 </li></ol>第3步。颅表面的预处理 （类似的程序应采用“直接充填修复”的牙科产品。） <ol><li>引物治疗<ol><li> 混合PRIMAR液与B一个碟子上。 </li><li> 申请颅骨表面用小刷子的混 ​​合物。 </li><li> 离开30秒。 </li><li> 干燥，温和的气流 。 （不洗。） </li></ol></li><li>粘接剂的应用<ol><li> 粘接剂涂抹于颅骨表面用小刷子。 </li><li> 应用柔和的气流 。 （不洗。） </li><li> 光固化20秒。 </li></ol></li></ol>第4步。安装复合树脂<ol><li>摩树脂与普通的宣传员 ，如果是一个普通的宣传员不可用，可以用来代替一个微型的锅铲 。 </li><li>光固化</li><li>增量安装 增量安装建议，以确保完整的树脂反应。 对于增量式安装，适用于每一个安装前的粘接剂 。 </li><li>模具的最终硬化和光固化复合树脂。 </li><li>手术结束</li></ol> 清洁手术区，适用于抗生素（庆大霉素药膏），并删除从立体火焰的动物。然后，在热控室（温度= 33-35℃）麻醉后恢复的动物。 在视频显示，无异常行为观察在任何动物手术后的第二天 。

<ol>
	<li>Adell, A., Artigas, F., Boulton, A., Baker, G., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brain+microdialysis:+Principles+and+applications.">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Kendrick, K., Robinson, T., Justice, J., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+large+unrestrained+animals:+neuroendocrine+and+behavioral+studies+of+acetylcholine,+amino+acid,+monoamine+and+neuropeptide+release+in+the+sheep.">Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep.</a> Microdialysis in the Neuroscience. 7, 327-348 (1991).</li><li>Oakley, B., Schafer, 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> Experimental Neurobiology: A laboratory Manual. , (1978).</li><li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li><li>Boulton, A., Baker, G., Robinson, T., Bateson, 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+relation+of+brain+electrical+activity+to+behavior.">The relation of brain electrical activity to behavior.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+finch.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch.</a> , 101-104 (2007).</li></ol>

根据动物实验的脑科学研究所，理化学研究所（RIKEN - BSI）和研究所的动物伦理委员会批准他们的指导方针进行了实验。

引进技术相对于传统的自固化丙烯酸树脂的应用，有几个重要的优点： 1。直接键合： 可以附着到骨自固化丙烯酸一段时间，但它不会坚持到骨。因此，锚固螺丝需要修复安装基座（见图2a）。相比之下，光固化树脂可以直接让螺丝不需要骨（图2B）的债券。因此，安装基座的大小，可以大大减少。此外，光固化树脂还坚持某些金属，如果你用“金属底漆”。粘接可变材料的这一特点，使我们一个...

1A照片显示微透析探针（一），螺帽（b）和指导导管（C）。在这张照片中，引导导管牙科树脂坚持头骨直接包围。照片B显示了一鸟在药物灌注。在头顶上，有一个微透析探针连接到罚款tefron管。比例尺= 1厘米，分别为A和B的2厘米，。 <img src="http://www.jove.com/files/249_2008_8_26_3/249_Figure-01.png" /> 图1 -一个微透析探针（A）和鸟在药物灌注的照片（二 ）

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

在慢性录音实验中，自固化牙科的丙烯酸树脂已被用来作为一个电极或微透析探针的安装基座。由于这些丙烯酸骨不债券，螺丝已被用来作为锚。然而，雀或鼠标一样的小实验动物，他们的头盖骨是很脆弱的，并不能成功举办的基石。在这份报告中，我们提出了一个新的应用程序安装电极或微透析探针在慢性实验基地牙科光固化树脂。这种材料可直接粘接头盖骨。因此，锚钉不需要手术视野，可以大大减少。以往的经验表明，粘接效果保持2个月以上。传统的树脂的窗口时，材料柔韧性和可行的几分钟。然而，这些牙齿胶粘剂的工作时间的窗口，是显着更广泛和可调。

Watch this Scientific Journal Video about Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments at JoVE.com

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

在这份报告中，我们提出了一个新的应用程序安装电极或微透析探针在慢性实验基地牙科光固化树脂。这种材料可直接粘接头盖骨。

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

application-light-cured-dental-adhesive-resin-for-mounting-electrodes

Research

JoVE Journal

Biology

15.3K 次观看.  RIKEN. 在这份报告中，我们提出了一个新的应用程序安装电极或微透析探针在慢性实验基地牙科光固化树脂。这种材料可直接粘接头盖骨。

视频: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

科研

生物学

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

诱导小鼠的牙周炎

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species&#39; normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

哺乳动物颅骨标本的牙齿和颞下颌关节病理学系统评估

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

建立牙髓干细胞的原代培养物

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

作者聚焦：使用牙髓干细胞推进组织再生和疾病建模

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

牙髓干细胞的原代培养

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...