The authors have no acknowledgements.

注:所有动物的程序都与辛辛那提大学的机构动物护理和使用委员会（IACUC），并按照该指南实验动物的护理和使用来自美国国立卫生研究院（NIH出版号的审批进行，85-23，1996年修订）。 1.安乐死和消毒鼠标<ol><li>鼠标放置在含有异氟醚的supratheraputic剂量的Dropbox，并允许吸气效果。一旦老鼠被安乐死，从包装盒中取出。 </li><li>与颈部脱臼安乐死的第二个手段。 </li><li>由清洁的动物用70％乙醇消毒鼠标的外表面上。 </li></ol> 2.识别和三种不同脂肪仓库隔离<ol><li>棕色脂肪组织（BAT）隔离: <ol><li>确保毛皮是由酒精清洗湿通过表皮和真皮Laye的削减，以帮助RS。 </li><li>将鼠标在其对表背仰卧位。 </li><li>抢皮肤正下方用钳子，电梯隔膜和切割用剪刀。 </li><li>切割横向围绕鼠标的圆周以露出腹膜。 </li><li>由脱套鼠标的上半部分显示蝴蝶形的BAT仓库。保持较低的附属物和腹部，一手提拉肌肤，迈向头。 </li><li>定向鼠标，以便它被定位俯卧在桌子上。要小心，不要污染暴露车厂的头发。 </li><li>洁净手术器械和改变一双崭新的手套。 </li><li>找到肩胛和相应的库。小心取出任何肤浅的白色脂肪之上蝴蝶，然后解剖的肩胛间棕色脂肪的蝴蝶。小心避免与棕色脂肪密切相关的肌肉。 注意:使用解剖显微镜，建议去除W的海特脂肪，以及在从肩胛褐色脂肪的分离。 </li><li>去除脂肪库，并转移到2 ml离心管。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </li></ol></li><li>皮下脂肪组织（SQ）的隔离，一个白色脂肪组织得宝（WAT）: <ol><li>穿上一双崭新的手套作为脂肪仓库之间没有交叉不纯物。 </li><li>露出腹股沟，三角SQ库由去手套革底部鼠标的一半。用于保持上附肢和胸部，一手拉动皮肤向下朝向脚用另一只手。 </li><li>定位鼠标在仰卧位时注意不要污染暴露车厂的头发。 </li><li>清洁surgica升仪器和改变一双崭新的手套。 </li><li>仔细剖析的SQ脂肪的三角形。要小心，不要污染样品肌肉，脂肪的邻国，乳腺或切割任何船只和污染的血液样本。 注意:使用解剖显微镜，建议如果边界不清晰。 </li><li>去除脂肪仓库和转让〜2毫升离心管。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。 </li><li>对于染色，固定或嵌入在十月如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </li></ol></li><li>隔离性腺脂肪内脏脂肪组织（VAT）和白色脂肪组织（WAT）车厂: <ol><li>穿上一双崭新的手套作为脂肪仓库之间没有交叉不纯物。 </li><li>脊髓损伤ssors，切开腹膜横向，正下方​​的隔膜。从隔膜到直肠中间剪开腹膜冠，露出腹部器官。 </li><li>定位睾丸或卵巢，并确定所连接的白色脂肪组织，称为附睾脂肪在雄性或性腺脂肪在女性。 </li><li>洁净手术器械和改变一双崭新的手套</li><li>仔细剖析从睾丸，附睾和输精管附睾两种脂肪库。或者，如果女性，小心地从卵巢解剖两种性腺脂肪垫。 </li><li>去除脂肪库，并将它们传送至2 ml离心管中。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。 </li><li>对于染色，固定或嵌入在十月如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </LI&gt; </ol></li></ol> 3.隔离血管周围脂肪组织（PVAT） <ol><li>孤立的心: <ol><li>莱鼠标在与向外扩展上下附属物仰卧位。 </li><li>安全附属物使用医用胶带。 </li><li>之后将鼠标上面列出，通过在剑突与钳提起制造紧张。水平切割通过隔膜，暴露胸腔的下部。 </li><li>同时维持张力，通过在剑突抬起，通过胸腔切开优朝向头部，只是到胸骨的一侧。 </li><li>拿起左肋只是次于锁骨，并沿着锁骨下长度切出朝向两个方向的腋下。胸腔和它的内容（心脏，肺等 ）现在应该清楚可见。 </li><li>通过使用无菌GA清洁胸外来​​血液和液体腔UZE吸收流体。如果收集器官或容器一定要喷出大量（补充信息）。 </li><li>一旦该区域被清除的液体，切支气管和血管连接以消除肺部，从而更好的暴露的心脏。 </li></ol></li><li>本地化和主动脉切除血管周围脂肪组织（PVAT）: <ol><li>删除以下机关更好地识别主动脉的下部分:肝，胃，脾，胰，肠和结肠癌。 </li><li>首先从识别胃和食管。切开食管的胃食管结合部，以释放从身体的腹部。 </li><li>其次，确定肠子和周围的肠系膜。切割表面通过肠系膜，因为它位于非常接近主动脉的肾部分，然后"运行肠"。 </li><li>切结肠释放尽量靠近直肠越好。因此，释放的胃，肠和结肠的鼠标。 </li><li>回复通过在附着肠系膜血管和切割移动胃，肠，结肠，胰腺和脾脏。胰腺和脾脏应该是免费的胃，小肠和结肠。 </li><li>通过削减通过肝静脉和肠系膜连接取出肝脏，删除所有叶。 </li><li>剪去内脏层和脂肪包围的肾脏。离开附加到主动脉体内肾脏作为地理标记主动脉的不同部分。 </li><li>冲洗用无菌1X PBS面积和吸收删除所有流体用无菌纱布。 </li><li>用微型剪刀和微型镊子，从它的背侧连接到脊椎和其腹侧附着到食道分离主动脉。 </li><li>按照和分离主动脉降主动脉的长度从原点的心脏在髂骨地区分叉隔离主动脉。 </li><li>识别并隔离锁骨下血管。隔离这些船只从脖子到主动脉根部，以更好地暴露在心脏主动脉结和根。 </li><li>取出胸腺然后切头臂动脉，左颈总动脉和左锁骨下动脉，使心脏的运动。 </li><li>用解剖显微镜的帮助下，查看围绕主动脉的血管周围脂肪组织（PVAT）层。 </li><li>采取非常谨慎，不捏或挤压PVAT，轻轻一拉PVAT方式，从微型钳主动脉。轻轻切PVAT的附接至与起始于胸部区域刚好优于其中隔膜位于微型剪刀主动脉。 注:解剖镜下建议。 </li><li>重复此过程主动脉的整个长度，在下腹主动脉区域，它位于正好优于主动脉血管的髂分叉精加工。 </li><li>如果主动脉弓PVAT需要，用同样的方法从较小的c取出PVATurvature拱。 </li><li>将PVAT样品分为2 ml离心管（S）。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 注意:如果有兴趣全面脂肪车厂分析更多的脂肪车厂考虑包括:腹膜后，肠系膜，大网膜，心包及腘。 </li></ol></li></ol>

<ol>
	<li>Ogden, C. L., Carroll, M. D., Kit, B. K., Flegal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+in+the+United+States,+2009-2010.">Prevalence of obesity in the United States, 2009-2010.</a> NCHS Data Brief. 82, 1-8 (2012).</li><li>Flegal, K. M., Carroll, M. D., Kit, B. K., Ogden, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+and+trends+in+the+distribution+of+body+mass+index+among+US+adults,+1999-2010.">Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999-2010.</a> 307 (5), 491-497 (2012).</li><li>Cawley, J., Meyerhoefer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medical+care+costs+of+obesity:+an+instrumental+variables+approach.">The medical care costs of obesity: an instrumental variables approach.</a> Journal of Health Economics. 31 (1), 219-230 (2012).</li><li>Marder, W., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+Obesity:+Costs,+Treatment+Patterns,+Disparities+in+Care,+and+Prevalent+Medical+Conditions.">Childhood Obesity: Costs, Treatment Patterns, Disparities in Care, and Prevalent Medical Conditions.</a> Thomson Medstat Research Brief. , (2006).</li><li>Cortez, M., Carmo, L. S., Rogero, M. M., Borelli, P., Fock, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fat+diet+increases+IL-1,+IL-6,+and+TNF-&#945;+production+by+increasing+NF-&#954;B+and+attenuating+PPAR-&#947;+expression+in+bone+marrow+mesenchymal+stem+cells.">A high-fat diet increases IL-1, IL-6, and TNF-&#945; production by increasing NF-&#954;B and attenuating PPAR-&#947; expression in bone marrow mesenchymal stem cells.</a> Inflammation. 36 (2), 379-386 (2013).</li><li>Sanchez-Gurmaches, J., Hung, C. M., Sparks, C. A., Tang, Y., Li, H., Guertin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+loss+in+the+Myf5+lineage+redistributes+body+fat+and+reveals+subsets+of+white+adipocytes+that+arise+from+Myf5+precursors.">PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors.</a> Cell Metab. 16 (3), 348-362 (2012).</li><li>Sethi, J. K., Vidal-Puig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+Adipose+tissue+function+and+plasticity+orchestrate+nutritional+adaptation.">Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation.</a> J Lipid Res. 48 (6), 1253-1262 (2007).</li><li>Aarsland, A., Chinkes, D., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+and+whole-body+fat+synthesis+in+humans+during+carbohydrate+overfeeding.">Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding.</a> Am J Clin Nutr. 65 (6), 1774-1782 (1997).</li><li>Park, A., Kim, W. K., Bae, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+of+white,+beige+and+brown+adipocytes+derived+from+mesenchymal+stem+cells.">Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells.</a> World J. Stem Cells. 6 (1), 33-42 (2014).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and.</a> 150 (2), 366-376 (2012).</li><li>Giralt, M., Villarroya, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White,+brown,+beige/brite:+different+adipose+cells+for+different+functions.">White, brown, beige/brite: different adipose cells for different functions.</a> Endocrinology. 154 (9), 2992-3000 (2013).</li><li>Casteilla, L., P&#233;nicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity.">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity.</a> Methods Mol. Biol. 456, 23-38 (2008).</li><li>Grant, R., Youm, Y. H., Ravussin, A., Dixit, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+adipose+tissue+leukocytosis+in+obesity.">Quantification of adipose tissue leukocytosis in obesity.</a> Methods Mol. Biol. 1040, 195-209 (2013).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

肥胖可能导致大的宿主疾病发病率和该脂肪起着尚不完全清楚的作用的充分理解;因此继续研究在脂肪的字段是必要的。动物模型，特别是小鼠模型非常适合在疾病和潜在的药物治疗试验的进展最初的研究。在使用这些模型，精确的隔离和脂肪车厂切除脂肪影响了疾病的病理研究中的一个非常重要和必要的工具。 在关于脂肪库目前的文献，有文献关于代谢行为和脂肪仓库之间?...

脂肪做了一个显着的出现在媒体的聚光灯下，由于在过去几十年的20世纪肥胖的急剧增加。目前，肥胖的影响超过三分之一的成年人和在美国（美国）1儿童和青少年的17％。跨越各族，周围肥胖的统计研究表明，非西班牙裔黑人有肥胖（49.5％）的最高年龄调整率与墨西哥裔美国人（40.4％），所有西班牙裔（39.1％），和非比较西班牙裔白人（34.3％），2。肥胖的经济效应也为医疗保健系统日益受到关注。在2012年，据估计，每年的医疗照顾，肥胖在美国，2005年费用为190.2十亿，近21％的整体医疗支出预算。可悲的是，儿童肥胖，估计是负责单独$ 14十亿的直接医疗费用。在统计上，它被确定的平均医疗费用肥胖人士是2741美元高于去年比没有这种发病率3-5。 肥胖是用于各种诸如条件的主要危险因素:2型糖尿病，血脂异常，心血管疾病，癌症，肌肉骨骼疾病和慢性炎症。肥胖是深深依赖于代谢综合征等慢性疾病6-8的发病机制。随着肥胖等合并症之间的这种激烈的负极连接，科研集中注意力，以便更好地了解当前的流行和脂肪所扮演的多样和重要的角色。 从历史上看，脂肪组织被认为是无关紧要的，并仅仅看作是一个简单的填充组织。目前，脂肪已被证明在体内的功能中发挥许多重要作用:代谢，激素调节，炎症，保护和绝缘9。脂肪组织主要由脂肪细胞还含有周细胞，血管内皮细胞，单核细胞，巨噬细胞和多能干细胞8。脂肪组织是分布在整个身体中不同的仓库。主体仓库可以皮下发现，皮下，肌肉内，和本能10。脂肪库已被证明有仓库特定的代谢概况，从而衍生出一个仓库特定易感性肥胖及相关病症8。 传统上，脂肪组织已被分为两种主要类型:白色脂肪组织（WAT）和棕色脂肪组织（BAT）;虽然最近的文献表明，第三组的派驻命名BRITE或米色脂肪11。脂肪组织已被证明具有不同的颜色，形态，代谢功能，生化特征和表达10的遗传模式。在WAT脂肪细胞有一个单一的，大的脂滴和线粒体的数量可变。 WAT在身体的皮下和内脏地方是显性找到。 WAT功能主要是作为能量储存和器官保护的部位。在BAT脂肪细胞有一个多房形态和丰富的线粒体。 BAT位于主要在颈部和胸部的大血管，以及肩胛12。 BAT主要功能是调节产热7能源花费的行为。 BRITE或米色脂肪已经显示的，共享一个类似的形态和表达BAT但已被发现是源自白色脂肪细胞11。 在这个手稿中描述的手术方法为研究人员提供的能力，分析不同效果的因素，如:环境，医药，和遗传学，对脂肪;以及有益或有害作用的脂肪起着病和总体健康状况。此外，提供了一种方法来鉴定和分离不同类型的脂肪组织，以以便更好地了解车厂之间的生化关系和差异。这可以在确定身体内的位置，功能，和类型的脂肪之间的关系提供帮助。该所述方法通过提供用于毛可视化，基因表达分析，蛋白质表达分析，组织学检查，以及初级细胞系的隔离体外研究实现这一目的。目前，有许多文章，提供深入了解不同脂肪库，以及它们的解剖位置的代谢行为;但不提供一个深入的方法，对如何具体定位，识别和隔离这些仓库。这种手术方法提供了精确的技术，其允许多个站的具有夹层和污染的最小量相比较，以设计用于一个或两个仓库13-14的分离方法更为隔离。 该协议的目的是提供精确的方法对不同类型的多个解剖位置脂肪库的鉴定和分离。

<table border="0" cellpadding="0" cellspacing="0" width="730">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Med-Vet International&#160;</td>
			<td>#RXISO-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">70% Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:125px;">07-678-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DMEF-12</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">D-6421</td>
			<td>Warm in waterbath before putting on tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">2 mL microcentrifuge tubes</td>
			<td style="width:175px;">Midsci</td>
			<td style="width:125px;">MCT-200-C-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Phosphate buffered saline</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">P5368-10PAK</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization, identification, and excision of murine adipose depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

识别和腹股沟皮下脂肪，肩胛间褐色脂肪，内脏附睾脂肪（ 图1），以及主动脉弓血管周围脂肪，胸主动脉脂肪，肾上主动脉脂肪和下腹主动脉脂肪的本地化（ 图2），使用取得了成功所描述的手术方法。 BAT和WAT样品之间的组织学检查和分化采用HE染色（H＆E）染色（ 图3）进行了积极评价。脂联素（ADIPOQ），过氧化物酶体增殖物激活受体γ（PPAR-γ），的RNA水?...

Watch this Scientific Journal Video about Localization, Identification, and Excision of Murine Adipose Depots at JoVE.com

Localization, Identification, and Excision of Murine Adipose Depots

由于肥胖等合并症之间的激烈负极连接，对脂肪的作用发挥的研究在疾病和整体健康是必要的。我们提出了一个协议，用于分离和脂肪仓库允许利用原位脂肪研究的切除和体外方法。

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

localization-identification-and-excision-of-murine-adipose-depots

Research

JoVE Journal

Medicine

40.9K 次观看.  University of Cincinnati College of Medicine. 由于肥胖等合并症之间的激烈负极连接，对脂肪的作用发挥的研究在疾病和整体健康是必要的。我们提出了一个协议，用于分离和脂肪仓库允许利用原位脂肪研究的切除和体外方法。 

视频: Localization, Identification, and Excision of Murine Adipose Depots

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 ...

科研

医学

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

隔离和小鼠主动脉切除;心血管疾病研究的一个多功能的技术

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

人类的褐色脂肪组织的仓库的正电子发射断层扫描/计算机断层扫描自动分段及注册磁共振图像

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

血管保留切除术与原发性吻合术

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

电磁导航经胸结节定位用于微创胸外科手术

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

慢性结肠炎相关结肠直肠癌的肿瘤切除术模型，通过卡卡贴片的精确定位

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...