Name: performing and processing fna of anterior fat pad for amyloid
Uploaded: 2010-10-30T17:00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

我们感谢邦妮Phetteplace，RN女士协助在图形FNAB过程的演示视频。

简介系统性淀粉样变性是一种高度可变的疾病，是指外，统称为淀粉样蛋白的各种蛋白质沉积。与β褶配置这些淀粉样纤维的沉积，导致各种临床表现取决于所涉及的器官。系统性淀粉样变性的临床相关的表现是有重要器官如心脏，肝脏，和/或肾的参与时指出。从历史上看，这些涉及机关将活检表现出的淀粉样蛋白的存在。这些温和的侵入性程序进行重大风险，包括出血。脂肪垫抽吸以来向人们展示了一个可靠，无创方法检测系统性淀粉样变性淀粉样蛋白在1。这个过程基本上与检索fibroadipose组织评估缺乏在小血管壁2淀粉样蛋白沉积在局部麻醉下于前腹壁皮下脂肪的吸脂。 淀粉样蛋白在组织鉴定的，最经常使用的方法仍然看到时，刚果红染色切片在偏振光显微镜3下可视化特征的苹果绿色双折射模式。当脂肪垫的愿望是，刚果红渍，可以做到无论是直接涂幻灯片或吸气脂肪组织的细胞块准备。然而，在早期阶段的淀粉样变性的患者有很少的淀粉样蛋白沉积，这大大降低了刚果红染色的细胞块切片4,5灵敏度。通过电子显微镜的脂肪垫抽吸的超微结构评价具有更好的可重复性，并提高了灵敏度 4 。因此，建议向所有为一个单元块的编制和执行电子显微镜 2脂肪垫抽吸。 脂肪垫的愿望是一个相对较低的成本和获得组织诊断系统性淀粉样变性的非侵入性的方法。本文介绍了脂肪垫抽吸过程，来样加工的细节，提交两个刚果红染色和电子显微镜超微结构评价标本。在这段视频中，我们展示了这种重复性好，简单的程序，以获取最佳的诊断材料。 1。执行前脂肪垫活检 A.麻醉，局部地区（图1和2） <ol><li>用酒精棉签或一个特定的机构首选的皮肤清洁剂，清洁皮肤，腹部下腹外侧向中线，脐下（图1）。 </li><li>标记一个约2 × 2英寸的菱形形状的区域，如图1所示，标记笔。 </li><li> 18G针吸约10毫升1％的利多卡因。将一个25G的1.5寸毫针注射器。移除窃听直立的注射器，同时按下活塞，直到液体配药，无气泡存在任何残留空气。 </li><li>麻醉沿线已标记的菱形区域，如图2所示的边界。开始，刚下在A点皮肤插入25G针，小心地推针，皮下点十，拉注射器推杆，以确保您不在范围内的船只。然后慢慢推柱塞，同时撤回针点，而不让皮肤针点（图2A3）一个渗透到利多卡因2.5毫升（约利多卡因¼ 10 mL注射器）。用针在皮肤下的改变对点的Y（图2A4）方向，推动皮下针点Y（图2B1）。一如以往，确认针是不是在船只和免除约2.5毫升利多卡因，一边慢慢退出通过点针一个（图2B4 2B3）。重复类似的步骤，从B点出发，从B点和配药利多卡因皮下X与B到Y（图2C1 2D4）。防止从扎伤出血和乙公司应用无菌衡量一块。 </li><li>您现在可以检查该地区是麻醉轻轻抚摸皮肤内麻醉菱形针尖/棉一块纱布一角，与相邻unanesthetized的皮肤比较。 </li></ol> B.执行脂肪垫抽吸术（图3） （应用局部麻醉前脂肪垫吸性能可能被绕过，根据区域和个人喜好。正确执行前脂肪垫抽吸FNAB程序可以在一个刺完成。然而，根据个别病人的痛阈，来回操纵18G针头在皮下脂肪组织相对distres唱。麻醉方法的描述，这里只有两个25G针头刺破达到的效果，并避免出现改善程序的耐受性疼痛。） <ol><li>到10毫升注射器组装18G 1.5寸毫针。装在注射器中的抓地力适当的应用程序和真空释放（“FNAB握/枪”）的注射器针头大会。 </li><li>针尖插入皮下脂肪内的清洗和麻醉的菱形面积（图3a）。 </li><li>完全缩回的注射器针头插入皮下组织产生真空（图3b）柱塞。 </li><li>保持真空和皮下脂肪（图3C通过3I）游刃于各个方向来回针。每个冲程应尽可能长时间而不让皮肤针来选择针的长度。最大采样是通过改变每一笔划的方向（图3I）。重要的是要保持切向浆膜和皮肤表面平行，以避免腹腔穿刺针的方向。 </li><li> fibroadipose组织聚集在注射器中。一旦足够fibroadipose组织碎片（碎片丰富的血液混合标本高达1毫升）进行检索，完全释放的真空，取出针（图3K）。 </li><li>有病人或助理申请程序方面的纱布垫，以防止血液外渗公司的压力。 </li></ol> 2。标本的处理<ol><li>放置在电子显微镜（图4B）戊二醛溶液至少5-6 fibroadipose组织碎片。 </li><li>根据机构的协议，fibroadipose组织碎片数涂片可能准备通过两个幻灯片之间（图4A）蔓延。 </li><li>剩余材料被允许血块在注射器（这可能需要5-7分钟，凝血时间而定）（图4C）。 </li><li>吸10％的福尔马林从墙上的注射器和自由浮动（图4C2）脱落，使凝结fibroadipose组织材料注射器。取下注射器的柱塞（图4C3）和凝结fibroadipose组织转移到10％福尔马林的容器开口端从注射器喷嘴端（图4C4）对面。 </li><li>适当标记的容器，并提交电​​子显微镜和H＆E节和刚果红染色偏光显微镜下的评价福尔马林戊二醛。如果涂片准备，他们可能会根据个别实验室的协议处理。 </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="图1" /> 图1。面积为麻醉前脂肪垫FNAB。 <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="图2" /> 图2。麻醉的局部地区。 <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="图3" /> 图3。执行前脂肪垫FNAB。 <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="图4" /> 图4。加工前脂肪垫吸提交实验室检测淀粉样蛋白沉积。 3。代表性的成果<img src="/files/ftp_upload/1747/1747fig5.jpg" alt="图5" /> 图5。淀粉样蛋白在小血管壁fibroadipose组织碎片。Fibroadipose组织嵌入福尔马林处理，石蜡，用刚果红染色，并通过偏光光学显微镜检查。淀粉样蛋白在小血管壁显示苹果绿双折射（白色箭头）。 <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="图6" /> 图6。缺乏组织淀粉样变性。苹果绿双折射缺席在不同的病人组织无淀粉样变性。蓝双折射的胶原纤维（白色箭头），通常存在于几乎所有的标本。 <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="图7" /> 图7。纤维与淀粉样蛋白在血管壁的是一致的。直，无分支的电子显微镜，随机分散的直径为8-10纳米的纤维，由淀粉样蛋白在血管壁的形成。

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

淀粉样变性的诊断通常是实现与受影响的器官，如肾脏，肝脏和/或心脏组织活检。然而，这种方法具有较高的诊断率，是侵入性和可能相关的并发症包括出血2。直肠，牙龈和骨髓活检诊断的首选方法在20世纪60年代6,7,8相对较少侵入。腹部脂肪垫抽吸据报道在1973年作为一种安全，微创，简单，更便宜的组织学诊断为系统性淀粉样变性 1程序。然而，检索到的标本处理的程序和方法的细节是不连贯报道。这视频和文章，描述过程中的步骤一步。 检测淀粉样蛋白在脂肪垫抽吸的方法，同样，也没有得到很好的一些研究比较和评估各种方法 1,5,6,7,8标准化。与刚果红染色前脂肪垫抽吸的评价是与观察者间的重复性较低，尤其是在淀粉样变性早期病例与缺乏淀粉样蛋白沉积5敏感。福尔马林固定石蜡包埋细胞块切片进行免疫组织化学和刚果红色荧光细胞学涂片进行报道，以提高诊断的灵敏度9,10。电子显微镜提高了脂肪垫抽吸4，5 fibroadipose组织小血管壁缺乏淀粉样纤维的检测和鉴定。根据这一信息，本文介绍了标本制备细胞块（刚果红染色细胞块切片在偏光显微镜诊断苹果绿色双折射下的评价，也表明免疫组化作为）和电子显微镜的处理。但是，根据淀粉样标本的评价选择的方法，它可能会受到细胞学涂片制备，细胞块的准备，处理和电子显微镜。有些实验室执行从吸气fibroadipose组织碎片，并准备与刚果红染色研究荧光 10涂片测试。 一般在后期的淀粉样变性，与刚果红偏光显微镜的光学显微镜，可能就足够了，但是，在疾病的刚果红染色与偏光显微镜对细胞块切片早期阶段是那么敏感和可靠的脂肪垫抽吸4,5,11 。 Thioflavin T等其他特殊污渍可使用类似的限制。其他的方法来检测淀粉样蛋白，包括与刚果红偏光显微镜和荧光 10淀粉样蛋白的吸涂片的评价。这种方法在我们的经验是少重现。通过电子显微镜淀粉样蛋白脂肪垫血管的评价克服了这些限制 4,11 。淀粉样蛋白的进一步鉴定已通过免疫电镜 12;然而，这些方法可能不会在非三级护理设置广泛。 用于执行共同愿望程序的针头可划分成细计的大小（21 - 25G），中间（18 - 20G），大（如14G）11 。前脂肪垫的愿望已报告使用变量仪表，从中间（18至20G）不等的罚款 ​​（21到22G）2,10执行。肿块大多是与更精细的表针（如25G）吸气，以取得良好的细胞学涂片检查，以及与更广泛的针头（如18G）来获取额外的样品细胞块的额外的通行证。 前脂肪垫抽吸期间，凝聚力的fibroadipose组织不吸以及更细的针头。细胞块的制备及电子显微镜是重要的评价小血在血管壁上的脂肪抽吸fibroadipose组织碎片。更宽的表针（ ​​如18G）推荐产生诊断提供充足的物质 2,10 。由于程序是可比常规FNAB的，被称为脂肪垫抽吸作为FNAB，尽管更广泛的表针 ，可用于执行5。

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

酒精棉签，纱布，标志着笔，10毫升注射器X2，1％利多卡因（局部麻醉剂，如果单独使用利多卡因，它会导致灌注后最初的烧灼感。这可以通过使用1:1混合的1％利多卡因和1％的钠双向碳酸盐），18号（G）的1.5英寸针X2（表演FNAB），25G 1 ½英寸针（注射局部麻醉剂），FNA的注射器持有人（“枪”），绷带，小瓶（电子显微镜），戊二醛和活检10％的福尔马林的容器。

performing and processing fna of anterior fat pad for amyloid

从历史上看，心脏，肝脏，肾脏活检，证明在淀粉样变性淀粉样蛋白沉积。由于本病的临床表现是如此的变量和非特异性，这些活组织切片检查相关的风险是太伟大的诊断率。其他网站有一个较低的活检的风险，如皮肤或牙龈，也相对侵入性和昂贵。此外，这些活组织切片检查可能并不总是有足够的淀粉样蛋白沉积，以建立一个诊断。脂肪垫的愿望表现出良好的临床相关性低的成本和最小的发病率。但是，有没有执行此过程或处理吸气标本，从而导致变量和nonreproducible结果的标准化协议。检测组织中淀粉样蛋白的最频繁使用的方​​式是对刚果红染色切片使用偏光显微镜的苹果绿色的双折射。这种技术需要细胞块吸气材料的制备。不幸的是，呈现在淀粉样变性的早期阶段的病人有极少量的淀粉样蛋白，从而大大降低了对刚果红染色细胞块切片的脂肪垫抽吸的敏感性。因此，应利用脂肪垫抽吸，通过电子显微镜超微结构评价，由于其增加了对淀粉样蛋白检测的灵敏度。本文演示了一个简单和可重复性的程序执行前脂肪垫抽吸利用细胞块切片和电子显微镜超微结构鉴定两个刚果红染色淀粉样蛋白的检测。

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

脂肪垫的愿望是首选，微创，成本低的方法，其他方法相比，检测诊断系统性淀粉样变性淀粉样蛋白。此视频文章演示了脂肪垫抽吸标本为最佳的诊断结果进行适当的处​​理与执行程序的轮廓。

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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Medicine

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

科研

医学

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

小鼠使用表面垫心电图心肌缺血再灌注损伤的确认

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

基质血管部分富集脂肪移植治疗症状性端 neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

痰液诱导和实验室处理方法

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

在体外进行血管重建的训练和测试系统

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...