这项工作是支持由美国国立卫生研究院R21 DC03969 - 01（日本国）。我们感谢弗里茨Lischka博士捕获RT - PCR检测显示在图4B和先生，贝利和S.女士阿拉尔孔技术援助执行单一的味蕾细胞，博士Tauf Huque。 M.亚尼纳Pepino是一个NIDAT32 DA07313华盛顿大学医学院，圣路易斯，MO（Cottler，LB PI）支持的一个老乡。

1。介绍和一般注意事项一般程序包括一个小的（1厘米2）面积的前舌背表面，去除一个（6-8）（味）的菌状乳头的数量，麻醉面积使用一个弯曲的弹簧微局部麻醉 - 剪刀（麦弗逊式Vannas类型＃简5603，Roboz，罗克维尔，医学博士）。删除的菌状乳头约0.25 毫米的 3个。有很少的非乳头（味道）去除上皮组织的每个乳头。新乳头的形式，在这一领域经过3至5个星期，恢复运作的味蕾（斯皮尔曼和品牌，未发表意见）。这少量的味道材料取出后的食物的味道或味道上有没有明显的效果。从个别成人人类的舌头的味蕾已经算的研究，可以预计，人类的舌头包含约5000个味蕾。我们的程序中删除这些味蕾大部分在20 25（平均3.5％至4菌状乳头味蕾在人力）2，或总数的约0.4％。此外，还有与这个组织的手术损失没有相关的疼痛。一个感觉，当然，仅在注射部位轻微疼痛，针进入舌头。麻醉消失后，不到一天的时间，偶尔轻度不适，但这种不适是比什么之一的经验后，意外地"咬"的舌头少得多。 在过去的几年中，我们已经开发了一个替代的过程，不包括麻醉。是两个相同的组织切除技术。在程序中的唯一的区别是，外科医生已不再局限于一个1厘米直径2地方的麻醉处理。而他/她可以选择突出的乳头出现舌背表面上的任何站点。删除乳头总数6-8，在这两种技术是有限的，如果正确执行或不麻醉，，取消6-8的菌状味道乳头的不适被认为是一个问题与管理相关的疼痛比较少一般调查的个人经历了两个程序，即局部麻醉，无需麻醉，结果显示，绝大多数科目将免除没有局部麻醉在今后所有的活组织切片检查。外科医生开发的这种技术（AIS）的，由作者进行活检无需麻醉授课的几位同事，都试图在自己和作证的不适是暂时的，被认为是小于的咬自己的舌头。最后，几十个人同意将活检，问他们是否愿意尝试去除没有麻醉的一个乳头，如果他们判断它是太痛苦了，他们将接受麻醉，都同意的情况下继续与去除乳头麻醉。 整个活检不应该需要更长的时间超过5-10分钟。如果采用麻醉另外2-3分钟。活检通常是在清晨进行。活检前，志愿者们指示预定开始时间前两小时吃一个便餐。活检，获得书面知情同意。活检时的排除标准包括那些患有严重的慢性疾病，是否用药或不控制（例如，任何胃肠道疾病，神经退行性疾病，肿瘤，血液恶液质，麻药过敏等），心率大于100或小于60，收缩压大于145或小于100，舒张压大于90或小于60，和外科医生的意见，可以参与这项研究的主题。 最后，重要的是要考虑在相同的设施，组织将检查处理，自组织的完整性正在迅速失去正如我们经常指出的mRNA质量下降，应进行活检。为了最大限度地减少这些问题的舌组织应尽快处理。 2。除准备<ol><li>主体是在牙科椅或头枕座位就座。 </li><li>检查活检时心率和血压。受血压，心率或口头的能力范围之外的下降，医生建议他/她看医生立即任命的主体。 </li><li>一般组织的健康检查口腔，注意口干舌，地域或任何舌病变的指标。口腔疾病的迹象显示，包括舌的病变或口干任何问题都排除在研究之外。指示须用清水冲洗口，免除所有的口头cavit碎片Y.如果有必要的舌头清洁一次性舌刮板清除牙菌斑或碎片，这可能是舌头在注射麻醉。在戴手套的手外科医生使用无菌纱布举行的舌头的前荐，因为它从口腔延伸。 </li><li>如果使用麻醉，与28计1CC结核菌素注射器x半"针肾上腺素（1:100000）含有0.25毫升利多卡因（2％）是慢慢注入到一个站点略低于舌头的前部分的背水面。远端收集站，以避免干扰与随后的研究利多卡因麻醉网站。相比之下，利多卡因注射量（0.25毫升）约一季度通常用于牙科手术的神经块（0.8毫升至1 mL）的金额。愿望后插入针，但注射麻醉剂前，确保没有血管袭击。这是采取预防措施，即使它在解剖学上不可能的大型船只将被击中的背水面。唯一的背水面上动脉，舌背动脉小血管主要位于后到前的三分之一，组织收割时的船只。较大的舌动脉和舌静脉，舌腹面上运行，远离背接受麻醉 3的面积。 1厘米麻醉的面积，似乎与周围组织相比略显苍白。这是由于组织，因为它压缩的小血管肿胀。这也是由于肾上腺素的缩血管作用。大约一分钟后，一个探头用于测试的麻木区。如果面积不麻木，第二次注射（0.1〜0.2毫升），可给予。 </li></ol> 3。舌乳头去除技术<ol><li>前季舌的主体是用无菌纱布包裹成（图1）和外科医生的左手（如果进行活检，用右手）举行。纱布的一角是开放的，允许外科医生擦拭唾液的舌头。舌头必须牢牢的，但是，当然，并非如此，造成不必要的不​​适。左手食指的尖端定位下的直接领导下进行活检网站舌。食指向上推，舌头变得拉长以上的手指使乳头坚持便于活检提示。 </li><li>选择乳头活检的标准:味道乳头filliform乳头周围很容易分辨，它们的形状和大小。菌状乳头是圆的，从周围有点孤立。在他们眼前的附近有一个圆形的空间，没有filliform乳头（图2A，蓝色箭头）。这个空间是直径约1毫米。菌状乳头的另一个显着特点是扩展的毛细血管分枝上最年轻的学科菌状乳头顶部可见。虽然树枝状定位菌状乳头更容易，他们不一定的，乳头中的味蕾存在的良好的预测。作为一项规则，开始走向前端的收获，并进一步远离舌尖，从模糊的未来组织收集的网站，以避免任何毛细血管出血。 </li><li>一旦一个很好的乳头已经确定，春季微型剪刀（麦弗逊式Vannas，5毫米，梳子，先端宽0.2毫米的总长度3"（RS - 5603，Roboz）是用来把它夹弯曲，锋利的尖端。为了有效地利用微型手术剪，不应使用切割刀片的一角;，而是尽量在刀片（图1，放大的图像，图3）尽可能接近的交点，与对面的刀片。去除乳头是实现用剪刀切割移动（图1）。应采取剪刀的整体状况，必须平行进一步针对性的乳头出现开放的刀片内，更有效的切割。舌背表面。 </li><li>有时候舌头会变得干燥，当医生是试图找出一个去除乳头。干燥后，运营​​商能够清楚地看到这是一个菌状乳头变得更具挑战性，在非常困难的乳头刀片滑动。过于干燥的舌头通常使金属刀片粘舌细腻的手术动作少可控。让主题接近他/她的嘴，并与舌表面接触唾液，有助于这一进程。过于潮湿的舌头也不好。在此过程中使用的纱布，用来存储舌角落帮助。 </li><li>确实发生任何毛细血管出血治疗用无菌纱布印迹。出血通常几个分钟内停止<html>犹特和不干预其他组织收割。由于乳头延长以上的舌头的上皮表面，只有一个菌状乳头上3 / 4被删除（图2B），这部分的味蕾菌状，出血少的预期。 </li><li>用一个小镊子杜蒙（模式类型），删除乳头被放置在冷冻基本林格氏缓冲液（氯化钠130毫米，1毫米氯化钙 ，氯化钾，5毫米1毫米氯化镁2，1毫米钠丙酮酸，20毫米的HEPES - NA ，pH值7.2〜305毫osmols渗透压为后续的操作）。切勿捏一个镊子刀片之间的乳头，这是势在必行。该组织是很脆弱的，并应特别小心处理。 </li><li>搬迁后的乳头，主体保持在10到15分钟的椅子上。每个主题被告知不咬其麻醉的舌头，但为了保持他们的舌头在休息。科目应视觉监控由主治外科医生。麻醉预计穿在大约30-60分钟。由于乳头拆除现场活检后可能会略有酸痛，指示他们要知道辛辣或热（温度）的食品，碳酸饮料或饮料，是极冷或极热。进一步表明，受试者的唾液，任何轻微的偏红的色彩是正常的，将血块溶解后停止。 </li><li>主题被视为评价的乳头被拆除现场的乳头上除名后的30-40天。这个时候的味道乳头已经完全再生。 </li></ol> 5。代表性的成果组织收集可用于免疫组织化学，RT - PCR技术， 原位杂交，钙成像，膜片钳和组织文化。当正确执行乳头的外观是健康的，白色，直径约0.5-1毫米（图2B）。 乳头进行免疫组化收集处理的基础上现有的和发布的协议4。一个典型的味蕾，从paraformaldehide固定的味道乳头如图4A所示。 活检乳头也被酶治疗5实现味蕾细胞分解。酶解后，细胞可维持在加湿的培养皿至4小时，在4C。在这种状态下，他们可用于单细胞PCR（图4B），钙成像等细胞显示出各种形状，许多细长和双极性（图5A和5B）正如人们所预料的味蕾细胞。当收集细胞进行钙成像，经过约20分钟，在显微镜下后，细胞开始凋亡滤过泡（图6A和6B，箭头），其中许多人成为一轮。在我们的实验室开发出一种技术，细胞拿起单独使用一个补丁移液器，图5A和5B所示。 如上所述，乳头开始5至8周后再生。图7A演示一个使用无需麻醉，从左侧删除志愿者的舌头8菌状乳头的舌头的外观。注意8轻微的淡红色斑点，乳头被剪断。无出血，手术后观察。本图像10分钟后活检。图7B是舌活检后40天看到同一主题的形象。请注意所有的乳头已经重新长出。要确定是否再生乳头功能，我们要求我们的志愿者同意第二次活检涉及完全相同乳头。要正确地确定适当的乳头，我们计划再收获，我们生成一个网格（图7C），适合8乳头被拆除摆在首位，后40天，最初活检后叠加在同一志愿者的舌头。借助舌头的轮廓和从边缘到电网的距离，一旦确定，我们已删除了几个完全相同的乳头和免疫组化处理他们。图4A是其中一个乳头。请注意一个味蕾（黄色）和磷脂Cβ2（PLCβ2），2型味觉细胞转导相关的酶免疫阳性染色的轮廓。这些数据表明，使用本文所述的活检，菌状乳头再生40天，充分也有可能功能的味蕾。 <img src="/files/ftp_upload/2201/2201fig1.jpg" alt="图1" /> 图1。前季度舌的主体是进入无菌纱布包裹，并在外科医生的左手举行。舌头必须坚守。在右边的放大的图像显示的剪刀和刀片乳头的位置适当的位置。 201/2201fig2.jpg"ALT ="图2"/&gt; 图2。 （一）菌状乳头带有蓝色箭头所示。这是有点孤立的圆形结构及其周围，并在其紧邻的是一个圆形的空间缺乏filliform乳头。 （二）菌状乳头微型剪刀秒后，它已被删除领略春天的一角。 <img src="/files/ftp_upload/2201/2201fig3.jpg" alt="图3" /> 图3。自活检使用一面镜子，没有麻醉的一个菌状乳头。插图说明剪刀的整体状况，必须与舌背表面平行。如果做得正确和atraumatically，麻醉放弃几乎所有的志愿者。 <img src="/files/ftp_upload/2201/2201fig4a.jpg" alt="图4a" /> 图4 A免疫组织化学的一个味蕾。一个菌状乳头可视化磷脂β2Nomarski使用抗体的光学和红色荧光蛋白标签。味蕾和几个标记的味觉细胞的轮廓显示为黄色。 <img src="/files/ftp_upload/2201/2201fig4b.jpg" alt="图4b" /> - PCR候选人酸味受体，酸敏感离子通道（ASIC1）B. RT。 M =标记。 1到4车道，单细胞RT - PCR方法从四个不同的志愿者的ASIC1。集合后，菌状乳头酶分离，细胞单独拿起ASIC1表达。泳道2和4 ASIC1的正常表达。 <img src="/files/ftp_upload/2201/2201fig5.jpg" alt="图5" /> 图5。 A.单味细胞图像拿起使用补丁吸管。一个大的上皮细胞是在左下角（5A）可见。两个红血细胞，也可见上述补丁吸管和后面的玻璃桶。味觉细胞是约30-50微米长，宽6微米左右。仅供参考，红血球直径约7-8微米。 B.显示了另一种味觉受体细胞，周围四个上皮细胞。味觉细胞看起来明快。 <img src="/files/ftp_upload/2201/2201fig6.jpg" alt="图6" /> 图6。答:图像的细胞开始研制凋亡滤过泡（鬼一样的味觉细胞，箭头的圆形滤泡）。 B.细胞不再收获时，其外观上的变化如下所示。 <img src="/files/ftp_upload/2201/2201fig7.jpg" alt="图7" /> 图7。 A.十几分钟后，8味道乳头不使用麻醉的去除。手术部位可见的略微泛红的斑点。注意到没有出血。 B.活检后40天的舌头。请注意所有的乳头再生。为了帮助确定删除重新长出乳头，与网站创建一个网格右侧的图片，并在左侧图像再生乳头转移。 C.再生乳头出现在完全相同的位置。收获乳头再次表明，再生的乳头载功能的味觉细胞（图4A）的免疫组织化学组。

<ol>
	<li>Breslin, P. A., Spector, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+taste+perception.">Mammalian taste perception.</a> Curr Biol. 26 (4), R148-R155 (2008).</li><li>Miller, I., Doty, R. I., Dekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+peripheral+taste+system.">Anatomy of the peripheral taste system.</a> Handbook of Olfaction and Gustation. , 521-547 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Figure+12.67.">Figure 12.67.</a> Gray's Anatomy: The Anatomical Basis of Medicine and Surgery. , (1995).</li><li>Huque, T., Cowart, B. J., Dankulich-Nagrudny, L., Pribitkin, E. A., Bayley, D. L., Spielman, A., Feldman, I., S, R., Mackler, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sour+ageusia+in+two+individuals+implicates+ion+channels+of+the+ASIC+and+PKD+families+in+human+sour+taste+perception+at+the+anterior+tongue.">Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.</a> PLoS One. 4 (10), e7347-e7347 (2009).</li><li>MacDonald, J. F., Mody, I., Salter, M. W., Spielman, A. I., Spielman, A. I., Brand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamping+of+taste+cells+in+mouse.">Patch-clamping of taste cells in mouse.</a> Experimental Cell Biology of Taste and Olfaction. , 329-332 (1995).</li></ol>

使用人体细胞的一个显着的优势是其特殊性。在这里详细介绍的过程提供了来自人类的味觉受体细胞的分离舌组织。这些味觉细胞，可用于分子生物学研究，试图发现味觉信息传导的重要分子，监测活动，以刺激味觉细胞的钙成像研究。此外，此过程中提供的舌组织可与各种技术， 如原位杂交，免疫组织化学分析，并允许捕捉如聚合酶链反应（PCR）技术，包括实时定量单细胞PCR的分...

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<td>McPherson-Vannas. Curved, sharp cutting edge 5mm. Comb, tip width 0.2 mm overall length 3"</td>
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<td>Roboz Surgical Instruments Co., Inc. Gaithersburg, MD 20898</td>
<td>RS-5603, Roboz</td>
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<td>Small Dumont forceps</td>
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<td>Roboz Surgical Instruments Co.</td>
<td>RS-4915, Roboz</td>
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technique to collect fungiform (taste) papillae from human tongue

味觉是人类生活的关键。通知将潜在摄入的食物质量的身体和刺激，准备消化道的消化代谢过程。对了解早期生物化学和分子事件的基本味道传导（检讨，布雷斯林和斯佩克特，2008年1）正在取得稳步进展。然而，迄今取得的进展在很大程度上导致动物模型。然而，由于在受体特异性和受体密度显着差异有所不同物种之间，人类的味道传导只会被理解，使用人类的味道组织。在这里，我们描述了一个活检技术，人类菌状乳头，可见圆形粉红色的前结构，收集包含味蕾直径约0.5毫米。这些切片乳头被用于多种用途，包括可行的味蕾细胞的分离 ，原位杂交，免疫组织化学，并通过分子生物学技术的，识别特定味道的新型蛋白质。

Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

Technique to Collect Fungiform Taste Papillae from Human Tongue

基本味觉转导的分子机制方面的知识最近享受到了重大进展，主要是由于使用动物模型。然而，在味觉的灵敏度和特异性哺乳动物认股权证在人体组织的研究广泛​​的多样性。我们描述了一个活检技术，收集从对人舌乳头的生活品位细胞。

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

technique-to-collect-fungiform-taste-papillae-from-human-tongue

Research

JoVE Journal

Medicine

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

28.8K 次观看.  College of Dentistry, New York University. 基本味觉转导的分子机制方面的知识最近享受到了重大进展，主要是由于使用动物模型。然而，在味觉的灵敏度和特异性哺乳动物认股权证在人体组织的研究广泛​​的多样性。我们描述了一个活检技术，收集从对人舌乳头的生活品位细胞。

视频: Technique to Collect Fungiform Taste Papillae from Human Tongue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

医学

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

简化技术<em&gt;原位</em&gt;切除角膜和视网膜组织剜人眼地球

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

使用层压技术来执行人类巩膜共聚焦显微镜


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

大鼠舌的机械和粘弹性的体内评估

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

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

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

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

小鼠横主动脉狭窄诱导左心室肥厚的技术研究

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

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

味觉考试: 一个简短的验证测试

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

主动脉瓣重置术治疗瓣膜备用主动脉根置换的规范化技术

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Psychophysical Tracking Method to Assess Taste Detection Thresholds in Children, Adolescents, and Adults: The Taste Detection Threshold (TDT) Test

This paper describes a two-alternative, forced-choice, staircase, tracking procedure, called the Taste Detection Threshold (TDT) test, that provides a reliable measure of sweet, salty, and umami taste detection thresholds from childhood to adulthood. Advantages of the method include procedures that are identical for children and adults, thus allowing the determination of age-related and individual differences in taste perception, if any, and tasks that can be completed in a relatively short time frame, do not rely on continuous attention or require memorization, control for subjective response biases, and minimize the impact of language development. After a 1 hour fast, participants are presented with pairs of solutions; in each pair, one solution is water, and the other solution contains varying concentrations of the tastant.
Using a whole-mouth tasting method, participants taste each solution (without swallowing and with rinsing between tastings) and then point to the solution with a taste or that tastes different from water. The concentration of the stimulus in the subsequent pair increases after a single incorrect response and decreases after two consecutive correct responses. A reversal occurs when the concentration sequence changes direction. The task is deemed completed after the occurrence of four reversals, provided there are a maximum of two dilution steps between two successive reversals, and the series of reversals do not form an ascending pattern. These additional criteria ensure greater reliability in outcomes. The TDT is then calculated as the geometric mean of the concentrations of the four reversals. This method has real-world relevance as it provides information on a dimension of taste perception that is independent of hedonics, and that can change with aging and certain disease states, making it a valuable psychophysical test.

Psychophysical Tracking Method to Assess Taste Detection Thresholds in Children, Adolescents, and Adults: The Taste Detection Threshold TDT Test

Psychophysical tools measure the functionality of the taste system for both research and health assessment purposes. This paper describes a method to measure taste detection thresholds that can determine the lowest concentration of sucrose, sodium chloride, or monosodium glutamate that can be tasted by individuals as young as 6 years.

评估儿童、青少年和成人味觉检测阈值的心理物理跟踪方法:味觉检测阈值（TDT）测试

This paper describes a two-alternative, forced-choice, staircase, tracking procedure, called the Taste Detection Threshold (TDT) test, that provides a ...