Name: technique to collect fungiform (taste) papillae from human tongue
Uploaded: 2010-09-18T17:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

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的分子程序的单个细胞。由于此过程可在人的味觉可以psychophysically评估活检前进行的，它是可以得到一个明确的负责味觉异常（例如，看到Huque等人，2009年）的分子细节的图片。这活检程序允许一个可行的和代谢完整的单细胞味蕾细胞丰富的准备，从而有能力执行这些细胞的膜片钳记录的生产。使用这种技术，未来的研究，不仅增加我们了解的味觉传导过程的独特的人，但也破译潜在的疾病或药物诱导的状态和发展方案来处理这些异常的味觉异常的分子机制。 最后，我们的观察结果表明，存在“漏洞”，常常注​​意到菌状乳头接近到arborizations，通常被认为是味蕾毛孔，事实上，不存在味蕾良好的预测。这些漏洞的性质究竟是什么，或者他们可能会执行什么功能，我们不知道。 在8年以来，我们开始此过程，我们已多次收集到4000多味乳头的数十名志愿者。两个不良的发病率，与局部麻醉相关领导的临时肿胀的舌头，但在未来48小时内内解决，没有任何不良影响。如果做得好，无菌和无创伤条件下，这种方法是安全有效的。

<table><tbody><tr><td>McPherson-Vannas. Curved, sharp cutting edge 5mm. Comb, tip width 0.2 mm overall length 3&quot;</td><td>Roboz Surgical Instruments Co.</td><td>RS-5603, Roboz</td><td></td></tr><tr><td>Small Dumont forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-4915, Roboz</td><td></td></tr></tbody></table>

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

28.9K 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

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

科研

癌症研究

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

神经科学

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

为菌状乳头的客观分析丹佛乳头协议

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

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 ...

医学

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

生物学

The Tongue and Taste Buds

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or contain the sensory taste (or gustatory) receptor cells (fungiform, circumvallate, and foliate papillae). Embedded within each taste-related papilla are the taste buds&mdash;clusters of 30 to 100 gustatory receptor cells.

Gustatory receptor cells extend finger-like projections called gustatory hairs (or microvilli) into a region known as the taste pore. Here, many of the cells contain receptors that detect different tastants&mdash;the molecules that can be tasted. The average number of taste buds varies significantly among individuals, with estimates ranging from 2,000-10,000 taste buds. Taste cells have a lifespan of about 10-14 days and are continually replaced. Thus, each taste bud contains taste cells at different stages of development.

<h4>Different Types of Taste Papillae</h4>

Aside from the filiform papillae, which do not contain taste buds, the mushroom-shaped fungiform papillae are the most numerous. Fungiform papillae are predominantly located on the anterior two-thirds of the tongue and contain between one and eight taste buds each.

In contrast, the other two types of papillae&mdash;circumvallate and foliate&mdash;contain more than 100 taste buds per papilla. Circumvallate papillae, the largest type, are located at the back of the tongue in &ldquo;V&rdquo;-like formation. Nearby, on the sides of the tongue, are the foliate papillae.

<h4>The Myth of the Tongue&rsquo;s Taste Map</h4>

The work of many scientists, including Collings, Yanagisawa, and colleagues, indicates that the five basic tastes can be perceived anywhere across the tongue. Thus, distinct tastes are not restricted to particular regions, as typically indicated on tongue maps.

舌头和味蕾

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or ...

教育

JoVE Core

营养与消化

肌肉骨骼系统

关键术语与概念

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

After euthanizing an adult mouse, use large sterile dissection scissors to cut the cheeks and break the jaw. Then, lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity. Cut the tongue out and collect it in sterile ice-cold dPBS with calcium and magnesium.
Under a dissecting microscope, remove and discard the anterior tongue by cutting just anterior of the intermolar eminence with a razor blade. Then, using a delicate task wipe to remove any hair and excess liquid from the posterior tongue.
Next, fill a 1-milliliter syringe with 200 to 300 microliters of injection enzyme solution and insert a 30-gauge, 1/2 inch needle just above the intermolar eminence until just interior of the circumvallate papillae or CVP. Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues. Withdraw the syringe slowly and continuously from the tongue as the solution is being injected.
Incubate the tongue in sterile calcium/magnesium-free dPBS at room temperature for precisely 33 minutes. Using extra fine dissection scissors, make small cuts in the epithelium bilaterally and just anterior of the CVP. Then, gently peel the epithelium by lifting it with fine forceps. Once the trench epithelium is free of the underlying connective tissue, place it in a 2-milliliter microcentrifuge tube pre-coated with FBS.

技术收集人舌菌状（味道）乳头

摘要

探索更多视频

此视频中的章节

舌细胞外基质的制备与舌鳞癌体外重建

人力蕈状味觉乳头细胞的分离和培养

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

为菌状乳头的客观分析丹佛乳头协议

味觉考试: 一个简短的验证测试

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

舌头和味蕾

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序

技术收集人舌菌状（味道）乳头

摘要

探索更多视频

此视频中的章节

舌细胞外基质的制备与舌鳞癌体外重建

人力蕈状味觉​​乳头细胞的分离和培养

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

为菌状乳头的客观分析丹佛乳头协议

味觉考试: 一个简短的验证测试

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

μTongue:一个基于微流体的功能成像平台，用于在Viva的舌头

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

舌头和味蕾

从小鼠模型中分离环状（CVP） 上皮:从小鼠舌中分离 CVP 上皮的酶促程序

人力蕈状味觉乳头细胞的分离和培养

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序